Compositions and methods for the depletion of CD117+ cells

ABSTRACT

The invention provides compositions and methods useful for the depletion of CD117+ cells and for the treatment of various hematopoietic diseases, metabolic disorders, cancers, e.g., acute myeloid leukemia (AML) and autoimmune diseases, among others. Described herein are antibodies, antigen-binding fragments, and conjugates thereof that can be applied to effect the treatment of these conditions, for instance, by depleting a population of CD117+ cells in a patient, such as a human. The compositions and methods described herein can be used to treat a disorder directly, for instance, by depleting a population of CD117+ cancer cells or autoimmune cells. The compositions and methods described herein can also be used to prepare a patient for hematopoietic stem cell transplant therapy and to improve the engraftment of hematopoietic stem cell transplants by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/576,572, filed on Oct. 24, 2017; U.S. Provisional Application No.62/596,569, filed on Dec. 8, 2017; U.S. Provisional Application No.62/632,967, filed on Feb. 20, 2018; and U.S. Provisional Application No.62/638,053, filed on Mar. 2, 2018. The contents of each of the priorityapplications is incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 23, 2018, isnamed 2018-10-23_Sequence_Listing_M103034_1100US.txt and is 211,836bytes in size.

FIELD OF THE INVENTION

The invention relates anti-CD117 antibodies, antibody drug conjugates(ADCs), and antigen-binding fragments thereof, as well as methods oftreating patients suffering from various pathologies, such as blooddiseases, metabolic disorders, cancers, and autoimmune diseases, amongothers, by administration of an antibody, or antibody drug conjugate(ADC), capable of binding an antigen expressed by a hematopoietic cell,such as a hematopoietic stem cell.

BACKGROUND OF THE INVENTION

Despite advances in the medicinal arts, there remains a demand fortreating pathologies of the hematopoietic system, such as diseases of aparticular blood cell, metabolic disorders, cancers, and autoimmuneconditions, among others. While hematopoietic stem cells havesignificant therapeutic potential, a limitation that has hindered theiruse in the clinic has been the difficulty associated with ensuringengraftment of hematopoietic stem cell transplants in a host.

There is currently a need for compositions that target specificendogenous stem cells that can be used as conditioning agents to promotethe engraftment of exogenous hematopoietic stem cell grafts such thatthe multi-potency and hematopoietic functionality of these cells ispreserved in the patient following transplantation.

CD117 (also referred to as c-kit or Stem Cell Factor Receptor (SCRF)) isa single transmembrane, receptor tyrosine kinase that binds the ligandStem Cell Factor (SCF). SCF induces homodimerization of cKIT whichactivates its tyrosine kinase activity and signals through both theP13-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998152(5):1259).

CD117 was initially discovered as an oncogene and has been studied inthe field of oncology (see, for example, Stankov et al. (2014) CurrPharm Des. 20(17):2849-80). An antibody drug conjugate (KTN0158)directed to CD117 is currently under investigation for the treatment ofrefractory gastrointestinal stromal tumors (GIST) (e.g., “KTN0158, ahumanized anti-KIT monoclonal antibody, demonstrates biologic activityagainst both normal and malignant canine mast cells” London et al.(2016) Clin Cancer Res DOI: 10.1158/1078-0432.CCR-16-2152).

CD117 is highly expressed on hematopoietic stem cells (HSCs). Thisexpression pattern makes CD117 a potential target for conditioningacross a broad range of diseases. There remains, however, a need foranti-CD117 based therapy that is effective for conditioning a patientfor transplantation, such as a bone marrow transplantation.

SUMMARY OF THE INVENTION

Described herein are antibodies, and antigen binding portions thereof,that specifically bind human CD117 (also known as c-kit), as well ascompositions and methods of using said antibodies. In particular, theantibodies and fragments described herein can be used in anti-CD117antibody drug conjugates (ADCs).

In one embodiment, the present invention provides compositions andmethods for the direct treatment of various disorders of thehematopoietic system, metabolic disorders, cancers, and autoimmunediseases, among others. The invention additionally features methods forconditioning a patient, such as a human patient, prior to receivinghematopoietic stem cell transplant therapy so as to promote theengraftment of hematopoietic stem cell grafts. The patient may be onethat is suffering from one or more blood disorders, such as ahemoglobinopathy or other hematopoietic pathology, and is thus in needof hematopoietic stem cell transplantation. As described herein,hematopoietic stem cells are capable of differentiating into a multitudeof cell types in the hematopoietic lineage, and can be administered to apatient in order to populate or re-populate a cell type that isdeficient in the patient. The invention features methods of treating apatient with antibodies and antibody drug conjugates (ADCs) capable ofbinding proteins expressed by hematopoietic cells, such as CD117(including, for example, GNNK+ CD117), so as to (i) directly treat adisease such as a blood disorder, metabolic disease, cancer, orautoimmune disease, among others described herein, by selectivelydepleting a population of cells that express CD117, such as an aberrantblood cell, cancer cell, or autoimmune cell, and/or (ii) deplete apopulation of endogenous hematopoietic stem cells within the patient.The former activity enables the direct treatment of a wide range ofdisorders associated with a cell of the hematopoietic lineage, as CD117may be expressed by a cancerous cell, such as a leukemic cell, anautoimmune lymphocyte, such as a T-cell that expresses a T-cell receptorthat cross-reacts with a self antigen, among other cell types. Thelatter activity, the selective depletion of hematopoietic stem cells, inturn creates a vacancy that can subsequently be filled bytransplantation of an exogenous (for instance, an autologous,allogeneic, or syngeneic) hematopoietic stem cell graft. The inventionthus provides methods of treating a variety of hematopoietic conditions,such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrichsyndrome, adenosine deaminase deficiency-severe combinedimmunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemiaand Schwachman-Diamond syndrome, human immunodeficiency virus infection,and acquired immune deficiency syndrome, as well as cancers andautoimmune diseases, among others.

In one aspect, the invention provides a method of depleting a populationof CD117+ cells in a human patient by administering an effective amountof an antibody, antigen-binding fragment thereof, capable of bindingCD117 where the antibody or fragment is conjugated to a cytotoxin(forming an ADC).

In another aspect, the invention provides a method of depleting apopulation of CD117+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD117conjugated to a cytotoxin (an ADC).

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD117 conjugated to a cytotoxin (forming an ADC) inan amount sufficient to deplete a population of CD117+ cells in thepatient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient an antibody orantigen-binding fragment thereof capable of binding CD117 conjugated toa cytotoxin (forming an ADC) in an amount sufficient to deplete apopulation of CD117+ cells in the patient, and subsequentlyadministering to the patient a transplant including hematopoietic stemcells.

In any of the above aspects, the cytotoxin may be, for example,Pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin, suchas α-amanitin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantthereof.

In some embodiments of any of the above aspects, the cytotoxin is anamatoxin or derivative thereof, such as α-amanitin, β-amanitin,γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinicacid, and proamanullin. In one embodiment, the cytotoxin is an amanitin.

In some embodiments of any of the above aspects, the cytotoxin is anamatoxin, and the antibody, or antigen-binding fragment thereof,conjugated to the cytotoxin is represented by the formula Ab-Z-L-Am,wherein Ab is the antibody, or antigen-binding fragment thereof, L is alinker, Z is a chemical moiety, and Am is the amatoxin. In someembodiments, the amatoxin is conjugated to a linker. In someembodiments, the amatoxin linker conjugate Am-L-Z is represented byformula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene; a dipeptide,—(C═O)—, a peptide, or a combination thereof; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117).

In some embodiments, Am contains exactly one R_(C) substituent.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

where S is a sulfur atom which represents the reactive substituentpresent within an antibody, or antigen-binding fragment thereof, thatbinds CD117 (e.g., from the —SH group of a cysteine residue).

In some embodiments, L-Z is

In some embodiments, Am-L-Z-Ab is one of:

wherein X is —S—, —S(O)—, or —SO₂—.

In some embodiments, Am-L-Z-Ab is:

In some embodiments, Am-L-Z-Ab is:

In some embodiments, Am-L-Z-Ab is:

In some embodiments, Am-L-Z is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof; Z is a chemical moietyformed from a coupling reaction between a reactive substituent presenton L and a reactive substituent present within an antibody, orantigen-binding fragment thereof, that binds CD117 (such as GNNK+CD117); and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

In some embodiments, L-Z is

In some embodiments, Am-L-Z is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, o a peptide, or a combination thereof;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117); and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form a 5-membered heterocycloalkylgroup of formula:

wherein Y is —(C═O)—, —(C═S)—, —(C═NR_(E))—, or —(CR_(E)R_(E′))—; and

R_(E) and R_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB), wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) and R_(D) are each as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ and R₅ are each independently H, OH, OR_(C), R_(C), or OR_(D);

R₆ and R₇ are each H;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H, OH, OR_(C), or R_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

In some embodiments, L-Z is

In some embodiments, Am-L-Z is represented by formula (II), formula(IIA), or formula (IIB)

wherein X is S, SO, or SO₂; R₁ is H or a linker covalently bound to theantibody or antigen-binding fragment thereof through a chemical moietyZ, formed from a coupling reaction between a reactive substituentpresent on the linker and a reactive substituent present within anantibody, or antigen-binding fragment thereof; and R₂ is H or a linkercovalently bound to the antibody or antigen-binding fragment thereofthrough a chemical moiety Z, formed from a coupling reaction between areactive substituent present on the linker and a reactive substituentpresent within an antibody, or antigen-binding fragment thereof; whereinwhen R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is the linker.

In some embodiments, the linker comprises a —(CH₂)_(n)— unit, where n isan integer from 2-6.

In some embodiments, R₁ is the linker and R₂ is H, and the linker andchemical moiety, together as L-Z, is

In some embodiments, Am-L-Z-Ab is:

In some embodiments, Am-L-Z-Ab is:

In some embodiments of any of the above aspects, the cytotoxin is amaytansinoid selected from the group consisting of DM1 and DM4. In someembodiments, the cytotoxin is an auristatin selected from the groupconsisting of monomethyl auristatin E and monomethyl auristatin F. Insome embodiments, the cytotoxin is an anthracycline selected from thegroup consisting of daunorubicin, doxorubicin, epirubicin, andidarubicin.

In another aspect, the invention features a method of depleting apopulation of CD117+ cells in a human patient by administering aneffective amount of an antibody, an antigen-binding fragment, or an ADCthereof capable of binding GNNK+ CD117.

In an additional, the invention features a method of depleting apopulation of CD117+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant containing hematopoietic stem cells, an effective amount ofan antibody or antigen-binding fragment thereof, or ADC capable ofbinding GNNK+ CD117.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantcontaining hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody, antigen-binding fragment thereof,or ADC capable of binding GNNK+ CD117 in an amount sufficient to depletea population of CD117+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient an antibody,antigen-binding fragment thereof, or ADC capable of binding GNNK+ CD117in an amount sufficient to deplete a population of CD117+ cells in thepatient, and subsequently administering to the patient a transplantincluding hematopoietic stem cells.

In some embodiments of any of the above aspects, the antibody,antigen-binding fragment thereof, is selected from the group consistingof a monoclonal antibody or antigen-binding fragment thereof, apolyclonal antibody or antigen-binding fragment thereof, a humanizedantibody or antigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv. In some embodiments, the antibody has anisotype selected from the group consisting of IgG, IgA, IgM, IgD, andIgE.

In some embodiments of any of the above aspects, the antibody, orantigen-binding fragment thereof, is internalized by a hematopoieticcell, such as a hematopoietic stem cell, cancer cell, or autoimmune cellfollowing administration to the patient. For instance, the antibody, orantigen-binding fragment thereof, or ADC may be internalized byhematopoietic stem cells, cancer cells, or autoimmune cells byreceptor-mediated endocytosis (e.g., upon binding to cell-surface CD117,such as GNNK+ CD117). In some embodiments, a cytotoxin covalently boundto the antibody or antigen-binding fragment thereof may be releasedintracellularly by chemical cleavage (for instance, by enzymatic ornon-specific cleavage of a linker described herein). The cytotoxin maythen access its intracellular target (such as the mitotic spindleapparatus, nuclear DNA, ribosomal RNA, or topoisomerases, among others)so as to promote the death of an endogenous hematopoietic cell, such asan endogenous hematopoietic stem cell prior to transplantation therapy,an endogenous cancer cell, or an endogenous autoimmune cell, amongothers.

In some embodiments of any of the above aspects, the antibody, orantigen-binding fragment thereof, or ADC is capable of promotingnecrosis of a hematopoietic cell, such as a hematopoietic stem cell,cancer cell, or autoimmune cell, among others. In some embodiments, theantibody or antigen-binding fragment thereof may promote the death of anendogenous hematopoietic stem cell prior to transplantation therapy, anendogenous cancer cell, or an endogenous autoimmune cell, among others,by recruiting one or more complement proteins, natural killer (NK)cells, macrophages, neutrophils, and/or eosinophils to the cell, such asa hematopoietic stem cell upon administration to the patient.

In some embodiments of any of the above aspects, the transplantcontaining hematopoietic stem cells is administered to the patient afterthe concentration of the antibody, antigen-binding fragment thereof, orADC has substantially cleared from the blood of the patient.

In some embodiments of any of the above aspects, the hematopoietic stemcells or progeny thereof maintain hematopoietic stem cell functionalpotential after two or more days (for example, from about 2 to about 5days, from about 2 to about 7 days, from about 2 to about 20 days, fromabout 2 to about 30 days, such as 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, or more) following transplantation of the hematopoietic stem cellsinto the patient.

In some embodiments of any of the above aspects, the hematopoietic stemcells or progeny thereof are capable of localizing to hematopoietictissue, such as the bone marrow, and/or reestablishing hematopoiesisfollowing transplantation of the hematopoietic stem cells into thepatient.

In some embodiments of any of the above aspects, upon transplantationinto the patient, the hematopoietic stem cells give rise to recovery ofa population of cells selected from the group consisting ofmegakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,myoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes,monocytes, osteoclasts, antigen-presenting cells, macrophages, dendriticcells, natural killer cells, T-lymphocytes, and B-lymphocytes.

In some embodiments of any of the above aspects, the method is used totreat one or more disorders, such as by depleting a population ofhematopoietic stem cells in a patient prior to hematopoietic stem celltransplant therapy so as to provide a niche to which the transplantedhematopoietic stem cells may home. Following transplantation, thehematopoietic stem cells may establish productive hematopoiesis, so asto replenish a deficient cell type in the patient or a cell type that isbeing actively killed or has been killed, for instance, bychemotherapeutic methods. For instance, the patient may be one that issuffering from a stem cell disorder. In some embodiments, the patient issuffering from a hemoglobinopathy disorder, such as sickle cell anemia,thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrichsyndrome. The patient may be suffering from an immunodeficiencydisorder, such as a congenital immunodeficiency disorder or an acquiredimmunodeficiency disorder (e.g., human immunodeficiency virus oracquired immune deficiency syndrome). In some embodiments, the patientis suffering from a metabolic disorder, such as glycogen storagediseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, and metachromatic leukodystrophy. In some embodiments,the patient is suffering from a disorder selected from the groupconsisting of adenosine deaminase deficiency and severe combinedimmunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, systemic sclerosis,systemic lupus erythematosus, and juvenile rheumatoid arthritis. In someembodiments, the patient is suffering from an autoimmune disease, suchas scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease,ant Type 1 diabetes. In some embodiments, the patient is suffering fromcancer or myeloproliferative disease, such as a hematological cancer. Insome embodiments, the patient is suffering from acute myeloid leukemia(AML), acute lymphoid leukemia, chronic myeloid leukemia, chroniclymphoid leukemia, multiple meloma, diffuse large B-cell lymphoma, ornon-Hodgkin's lymphoma. In some embodiments, the patient is sufferingfrom a myelodysplastic disease, such as myelodysplastic syndrome.

In some embodiments of any of the above aspects, the method is used todirectly treat a cancer, such as a cancer characterized by CD117+ cells(e.g., a leukemia characterized by CD117+ cells), by administration ofan antibody, or antigen-binding fragment thereof, or ADC that depletes apopulation of CD117+ cancer cells in the patient and/or byadministration of an antibody, or antigen-binding fragment thereof, soas to deplete a population of endogenous hematopoietic stem cells priorto hematopoietic stem cell transplantation. In the latter case, thetransplantation may in turn re-constitute, for example, a population ofcells depleted during the process of eradicating cancer cells. Thecancer may be a hematological cancer, such as acute myeloid leukemia,acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoidleukemia, multiple meloma, diffuse large B-cell lymphoma, ornon-Hodgkin's lymphoma.

In some embodiments of any of the above aspects, the method is used totreat an autoimmune disease, such as by administration of an antibody,antigen-binding fragment thereof, or ADC so as to deplete a populationof CD117+ autoimmune cells and/or by administration of an antibody, orantigen-binding fragment thereof, or ADC so as to deplete a populationof endogenous hematopoietic stem cells prior to hematopoietic stem celltransplantation. In the latter case, the transplantation may in turnre-constitute, for example, a population of cells depleted during theprocess of eradicating autoimmune cells. The autoimmune disease may be,for example, scleroderma, multiple sclerosis (MS), human systemic lupus(SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD),treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes), acutedisseminated encephalomyelitis (ADEM), Addison's disease, alopeciauniversalis, ankylosing spondylitisis, antiphospholipid antibodysyndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balodisease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas'disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Crohn's disease, cicatricalpemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinindisease, CREST syndrome, Degos disease, discoid lupus, dysautonomia,endometriosis, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease,Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, Hidradenitissuppurativa, idiopathic and/or acute thrombocytopenic purpura,idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis,juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease,Meniere disease, mixed connective tissue disease (MCTD), myastheniagravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), opticneuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,polychondritis, polymyositis and dermatomyositis, primary biliarycirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgiarheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter'ssyndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome,stiff person syndrome, Takayasu's arteritis, temporal arteritis (alsoknown as “giant cell arteritis”), ulcerative colitis, uveitis,vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), and Wegener'sgranulomatosis.

Thus, in some embodiments of any of the above aspects, the inventionfeatures a method of treating a hemoglobinopathy disorder, such assickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, andWiskott-Aldrich syndrome. In some embodiments, the invention features amethod of treating an immunodeficiency disorder, such as a congenitalimmunodeficiency disorder or an acquired immunodeficiency disorder(e.g., human immunodeficiency virus or acquired immune deficiencysyndrome). In some embodiments, the invention features a method oftreating a metabolic disorder, such as glycogen storage diseases,mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, and metachromatic leukodystrophy. In some embodiments,the invention features a method of treating a disorder selected from thegroup consisting of adenosine deaminase deficiency and severe combinedimmunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, systemic sclerosis,systemic lupus erythematosus, and juvenile rheumatoid arthritis In someembodiments, the invention features a method of treating an autoimmunedisease, such as scleroderma, multiple sclerosis, ulcerative colitis,Chron's disease, ant Type 1 diabetes. In some embodiments, the inventionfeatures a method of treating a cancer or myeloproliferative disease,such as a hematological cancer. In some embodiments, the inventionfeatures a method of treating acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymohoid leukemia, multiplemeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Insome embodiments, the patient is suffering from a myelodyplasticdisease, such as myelodysplastic syndrome. In these embodiments, themethod may include the steps of administering an antibody, orantigen-binding fragment thereof, or ADC that binds CD117 (e.g., GNNK+CD117) and/or a hematopoietic stem cell transplant according to themethod of any of the above-described aspects and embodiments of theinvention.

Similarly, in some embodiments of any of the above aspects, theinvention provides a method of treating cancer directly, such as acancer characterized by CD117+ cells (e.g., a leukemia characterized byCD117+ cells). In these embodiments, the method includes administeringan antibody, antigen-binding fragment thereof, or ADC that binds CD117(e.g., GNNK+ CD117). The cancer may be a hematological cancer, such asacute myeloid leukemia, acute lymphoid leukemia, chronic myeloidleukemia, chronic lymohoid leukemia, multiple meloma, diffuse largeB-cell lymphoma, or non-Hodgkin's lymphoma.

Additionally, in some embodiments of any of the above aspects, theinvention provides a method of treating an autoimmune disease, such asmultiple sclerosis (MS), human systemic lupus (SLE), rheumatoidarthritis (RA), inflammatory bowel disease (IBD), treating psoriasis,Type 1 diabetes mellitus (Type 1 diabetes) acute disseminatedencephalomyelitis (ADEM), Addison's disease, alopecia universalis,ankylosing spondylitisis, antiphospholipid antibody syndrome (APS),aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease (AIED), autoimmune lymphoproliferativesyndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease,bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliacsprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome,Degos disease, discoid lupus, dysautonomia, endometriosis, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture'ssyndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto'sthyroiditis, Hidradenitis suppurativa, idiopathic and/or acutethrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy,interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichenplanus, Lyme disease, Meniere disease, mixed connective tissue disease(MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome(OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, perniciousanemia, polychondritis, polymyositis and dermatomyositis, primarybiliary cirrhosis, polyarteritis nodosa, polyglandular syndromes,polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon,Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren'ssyndrome, stiff person syndrome, Takayasu's arteritis, temporalarteritis (also known as “giant cell arteritis”), ulcerative colitis,uveitis, vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), andWegener's granulomatosis. In these embodiments, the method includesadministering an antibody, or antigen-binding fragment thereof, or ADCthat binds CD117 (e.g., GNNK+ CD117).

In another aspect, the invention features a method of depleting apopulation of CD117+ (e.g., GNNK+ CD117+) cells by contacting thepopulation with an effective amount of a conjugate (or ADC) representedby the formula Ab-Z-L-Am, wherein Ab is an antibody or antigen-bindingfragment thereof that binds CD117, Z is a chemical moiety, L is a linkerand Am is an amatoxin. Am-L-Z may be represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocycloalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am contains exactly one R_(C) substituent.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

In some embodiments, L-Z is

In some embodiments, Am-L-Z is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117); and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

In some embodiments, L-Z is

In some embodiments, Am-L-Z-Ab is selected from

wherein X is —S—, —S(O)—, or —SO₂—.

In some embodiments, Am-L-Z-Ab is

In some embodiments, Am-L-Z-Ab is

In some embodiments, Am-L-Z-Ab is

In another aspect, the invention features a conjugate represented by theformula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD117 (e.g., GNNK+ CD117) and Am is an amatoxin. Insome embodiments, Am-L-Z is represented by formula (I), (IA), (IB),(II), (IIA), (IIB), (IV), (IVA), or (IVB), above.

In some embodiments, the antibody or antigen-binding fragment thereof isconjugated to the amatoxin by way of a cysteine residue in the Fc domainof the antibody or antigen-binding fragment thereof. In someembodiments, the cysteine residue is introduced by way of a mutation inthe Fc domain of the antibody or antigen-binding fragment thereof. Forinstance, the cysteine residue may be selected from the group consistingof Cys118, Cys239, and Cys265.

In some embodiments of these aspects, the cysteine residue is naturallyoccurring in the Fc domain of the antibody or antigen-binding fragmentthereof. For instance, the Fc domain may be an IgG Fc domain, such as ahuman IgG1 Fc domain, and the cysteine residue may be selected from thegroup consisting of Cys261, Csy321, Cys367, and Cys425.

In some embodiments of these aspects, R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

X is —S—, —S(O)—, or —SO₂—. In some embodiments, R₁ and R₂ are eachindependently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

X is —S—, —S(O)—, or —SO₂—. In some embodiments, R₁ and R₂ are eachindependently H or OH;

R₃, R₆, and R₇ are each H;

R₄ is OR_(C), or R_(C);

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

X is —S—, —S(O)—, or —SO₂—. In some embodiments, R₁ and R₂ are eachindependently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OR_(C) or NHR_(C);

R₉ is H or OH; and

X is —S—, —S(O)—, or —SO₂—. In some embodiments of these aspects, theantibody or antigen-binding fragment thereof is internalized by a CD117+cell.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD117 with a K_(d) of less than 1 μM, less than750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10nM, less than 1 nM, less than 0.1 nM, less than 10 μM, less than 1 μM,or less than 0.1 μM. In some embodiments, the K_(d) is from about 0.1 μMto about 1 μM.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD117 with a k_(ON) of from about 9×10⁻² M⁻¹ s⁻¹to about 1×10²M⁻¹s⁻¹.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD117 to a secondantibody or antigen binding fragment thereof, or binds the same epitopeas a second antibody, wherein the second antibody or antigen-bindingfragment thereof has the following complementarity determining regions(CDRs):

-   a CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1);-   a CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID    NO: 2);-   a CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:    3);-   a CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4);-   a CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and-   a CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6).

The disclosure further provides isolated anti-CD117 antibodies that maybe used in the antibody drug conjugates (ADCs) disclosed hereincomprising heavy and light chain CDRs and variable regions described inTable 1, Table 6, or Table 8.

In one embodiment, the anti-CD117 antibody, or antigen binding portionthereof, comprises a heavy chain variable region as set forth in theamino acid sequence of SEQ ID NO: 7, and a light chain variable regionas set forth in the amino acid sequence of SEQ ID NO: 8. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 9. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 11. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 12. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:13. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 14. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 15. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 17. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 18. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:19. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 20. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 21. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 23. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 24, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 25. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 26, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:27. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 28, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 29. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 30, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 31. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 32, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 33. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:34, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 35. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 36, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 37. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 38, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:39. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 40, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 41. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 32, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 42. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 43, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 44. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:45, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 46. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 47, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 48. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 49, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:50. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 51, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 52. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 53, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 54. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 55, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 56. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:57, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 58. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 59, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 60. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 61, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:50. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 62, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 63. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 64, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 65. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 66, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 67. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:68, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 69. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 70, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 71. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 72, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:73. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 74, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 75. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 76, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 77. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 78, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 79. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:80, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 81. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 82, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 83. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 84, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:85. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 86, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 87. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 88. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 89. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 90. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 91. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:92. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 94. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO:95. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 96. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 97. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:97. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 143, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 144. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 151, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 152. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 143, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 156. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:159, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 156. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 160, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 152. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 98, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:99. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 99. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 100. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 98, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 101. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:98, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 102.

In some embodiments of these aspects, the antibody, or antigen-bindingfragment thereof, is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV. In one embodiment, the antibody is anintact antibody.

In another aspect, the invention features a conjugate (ADC) representedby the formula Ab-Z-L-Cy, wherein Ab is an antibody or antigen-bindingfragment thereof that binds CD117 (e.g., GNNK+ CD117) and Cy is acytotoxin. In some embodiments of this aspect, the cytotoxin isPseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In some embodiments of this aspect, the antibody, antigen-bindingfragment thereof, or ADC is internalized by a CD117+ cell.

In some embodiments of this aspect, the antibody, antigen-bindingfragment thereof, or ADC binds CD117 with a K_(d) of less than 1 μM,less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM,less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM,less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 μM, lessthan 1 μM, or less than 0.1 μM measured by bio-layer interferometry(BLI). In some embodiments, the K_(d) is from about 0.1 μM to about 1μM.

In some embodiments of this aspect, the antibody, antigen-bindingfragment thereof, or ADC binds CD117 with a k_(on) of from about 9×10⁻²M⁻¹ s⁻¹ to about 1×10²M⁻¹s⁻¹ measured by a bio-layer interferometry(BLI) assay.

In certain embodiments, an anti-CD117 antibody, or antigen bindingfragment thereof, has a certain dissociation rate which is particularlyadvantageous when used as a part of a conjugate. For example, ananti-CD117 antibody has, in certain embodiments, an off rate constant(Kdis) for human CD117 of 1×10⁻² to 1×10⁻⁷, 1×10⁻³ to 1×10⁻⁷, 1×10⁻⁴ to1×10⁻⁷, 1×10⁻⁵ to 1×10⁻⁷, or 1×10⁻⁶ to 1×10⁻⁷ s⁻¹, measured by bio-layerinterferometry (BLI).

In some embodiments of this aspect, the antibody, antigen-bindingfragment thereof, or ADC competitively inhibits the binding of CD117 toa second antibody or antigen binding fragment thereof, wherein thesecond antibody or antigen-binding fragment thereof has the followingCDRs:

-   a CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1);-   a CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID    NO: 2);-   a CD-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:    3);-   a CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4);-   a CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and-   a CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6).

In some embodiments of this aspect, the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv. In some embodiments, the antibody has anisotype selected from the group consisting of IgG, IgA, IgM, IgD, andIgE.

In certain embodiments, the foregoing methods and compositions includean isolated anti-CD117 antibody or antigen-binding fragment thereofcomprising the CDRs set forth in the heavy and light chain amino acidsequences set forth in Table 1, Table 6, or Table 8. In certainembodiments, the foregoing methods and compositions include ananti-CD117 antibody or antigen-binding fragment thereof comprising thevariable regions set forth in the heavy and light chain amino acidsequences set forth in Table 1. In certain embodiments, the foregoingmethods and compositions include an IgG1 anti-CD117 antibody orantigen-binding fragment thereof comprises the variable regions setforth in the heavy and light chain amino acid sequences set forth inTable 1, Table 6, or Table 8.

In another aspect, the invention features a method treating acutemyeloid leukemia (AML) in a human patient, the method comprisingadministering an effective amount of an anti-CD117 ADC to the humanpatient such that AML is treated, wherein the anti-CD117 ADC comprisesan anti-CD117 antibody conjugated to a cytotoxin. In some embodiments,the cytotoxin is an RNA polymerase inhibitor. In another embodiment, theRNA polymerase inhibitor is an amatoxin. In other embodiments,anti-CD117 antibody comprises the CDR sequences set forth in the heavyand light chain amino acid sequences of Table 1. In yet otherembodiments, the anti-CD117 antibody is an intact antibody. In anotherembodiment, the antibody is an IgG1 or an IgG4.

In another aspect, the invention provides a conjugate represented by theformula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD117, L is a linker, Z is a chemical moiety, and Amis an amatoxin, wherein the antibody or antigen-binding fragment thereofcomprises the CDRs set forth in the heavy and light chain amino acidsequences of Table 1, Table 6 or Table 8.

In some embodiments, linker-chemical moiety-amatoxin portion (Am-L-Z) ofthe conjugate is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted C₁-C₆ alkyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

L is optionally substituted C₁-C₆ alkylene, optionally substituted C₁-C₆heteroalkylene, optionally substituted C₂-C₆ alkenylene, optionallysubstituted C₂-C₆ heteroalkenylene, optionally substituted C₂-C₆alkynylene, optionally substituted C₂-C₆ heteroalkynylene, optionallysubstituted cycloalkylene, optionally substituted heterocycloalkylene,optionally substituted arylene, optionally substituted heteroarylene, adipeptide, —(C═O)—, a peptide, or a combination thereof; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am comprises exactly one R_(C) substituent.

In some embodiments, L-Z is

In some embodiments, Am-L-Z is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted C₁-C₆ alkyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

L is optionally substituted C₁-C₆ alkylene, optionally substituted C₁-C₆heteroalkylene, optionally substituted C₂-C₆ alkenylene, optionallysubstituted C₂-C₆ heteroalkenylene, optionally substituted C₂-C₆alkynylene, optionally substituted C₂-C₆ heteroalkynylene, optionallysubstituted cycloalkylene, optionally substituted heterocycloalkylene,optionally substituted arylene, optionally substituted heteroarylene, adipeptide, —(C═O)—, a peptide, or a combination thereof; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am comprises exactly one R_(C) substituent.

In some embodiments, L-Z is

In some embodiments, linker-chemical moiety-amatoxin portion (Am-L-Z) ofthe conjugate is one of:

wherein X is —S—, —S(O)—, or —SO₂—, and Ab is shown to indicate thepoint of attachment of the antibody.

In some embodiments, linker-chemical moiety-amatoxin portion (Am-L-Z) ofthe conjugate is

wherein Ab is shown to indicate the point of attachment of the antibody.

In some embodiments, linker-chemical moiety-amatoxin portion (Am-L-Z) ofthe conjugate is

wherein Ab is shown to indicate the point of attachment of the antibody.

In some embodiments, linker-chemical moiety-amatoxin portion (Am-L-Z) ofthe conjugate is

wherein Ab is shown to indicate the point of attachment of the antibody.

In some embodiments, the Am-L-Z precursor is

wherein the maleimide reacts with a thiol group found on a cysteine inthe antibody.

In some embodiments, the Am-L-Z precursor is

wherein the maleimide reacts with a thiol group found on a cysteine inthe antibody.

In some embodiments, Am-L-Z is represented by formula (II), formula(IIA), or formula (IIB)

wherein X is S, SO, or SO₂;

R₁ is H or a linker covalently bound to the antibody or antigen-bindingfragment thereof through a chemical moiety Z, formed from a couplingreaction between a reactive substituent present on the linker and areactive substituent present within an antibody, or antigen-bindingfragment thereof; and

R₂ is H or a linker covalently bound to the antibody or antigen-bindingfragment thereof through a chemical moiety Z, formed from a couplingreaction between a reactive substituent present on the linker and areactive substituent present within an antibody, or antigen-bindingfragment thereof;

wherein when R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is thelinker.

In some embodiments, L-Z is

In some embodiments, Ab-Z-L-Am is

In some embodiments, Ab-Z-L-Am is

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the measurement of binding by Bio-LayerInterferometry (BLI) of the indicated purified IgG (sensor-associated)to purified human CD117 ectodomain (R&D Systems #332-SR) atconcentrations of 33.3 nM (top traces) and 11 nM (bottom traces) as afunction of time. The purified IgGs correspond to Ab1 (i.e., 001), Ab2(i.e., 002), Ab3 (i.e., 003), Ab4 (i.e., 004), Ab5 (i.e., 005), Ab6(i.e., 006), Ab7 (i.e., 007), Ab8 (i.e., 008), Ab9 (i.e., 009), Ab10(i.e., 010), Ab11 (i.e., 011), Ab12 (i.e., 012), Ab13 (i.e., 013), Ab14(i.e., 014), Ab15 (i.e., 015), and Ab16 (i.e., 016).

FIGS. 2A and 2B graphically depict the results of in vitro cell killingassays that show Kasumi-1 cell viability as measured in luminescene(RLU) by Celltiter Glo (FIG. 2A) or viable CD34+ CD90+ cell count (FIG.2B) in the presence of CD117-ADC or controls (y-axis) as a function ofCD117-ADC or controls concentration (x-axis).

FIGS. 3A, 3B, 3C and 3D graphically depict the results of an in vivocell depletion assay that shows CD117-ADC selectively depletes humanHSCs in humanized NSG mice. (A) Schematic of in vivo mouse model. (B)Shows the percent of human myeloid or (C) the percentage of T cellspresent in the peripheral blood of CD117-ADC or control treated mice,expressed as a percent of that cell population prior to treatment(normalized to baseline). (D) Shows the absolute number of CD34+ cellsin the bone marrow of CD117-ADC or control treated mice 21 days after asingle administration of the ADC.

FIGS. 4A, 4B, 4C and 4D graphically depict the results of an in vivotumor study that show CD117-ADC effectively depletes human leukemiccells in NSG mice. (A) Phenotypic analysis of Kasumi-1 cells in culture.(B) Phenotypic analysis of Kasumi-1 cells from bone marrow of tumorbearing mice at time of euthanasia. (C) Survival curve of mice treated 7days after tumor injection with CD117-ADC or a naked anti-CD117 antibodyor untreated controls. (D) Survival curve of mice treated 42 days aftertumor injection with CD117-ADC or a naked anti-CD117 antibody oruntreated controls.

FIGS. 5A, 5B and 5C graphically depict the results of an in vivo celldepletion assay that shows CD117-ADC selectively depletes HSCs in B6mice. (A) Schematic of an in vivo depletion assay in a B6 mouse model.(B) Shows the results of a mouse HSC depletion assay after theadministration of a single dose of an anti-CD117-ADC (i.e.,“CD117-Saporin”) in comparison to controls (i.e., “control” and“isotype-Saporin”), expressed as the kit+SCA+CD150+CD48− cell number(y-axis) as a function of a single injection of anti-CD117-ADC (i.e.,“CD117-Saporin”) or the controls (x-axis). (C) Shows the results of anengraftment assay, expressed as the percent donor chimerism (y-axis) asa function of treatment mode (i.e., “control,” “isotype-Saporin” and“CD117-Saporin”).

FIG. 6 graphically depicts the results of a non-human primatepharmacokinetic assay expressed as the concentration (ng/mL) of anisotype control antibody (i.e., “wild type antibody”) in comparison toan CK6 variant antibody with a shorter half life as a function time(i.e., hours post-administration; x-axis).

FIGS. 7A and 7B graphically depict the results of an in vivo depletionassay showing that CD117-ADC effectively depletes human CD34+ cells inhumanized NSG mice. (A) Phenotypic analysis of human cells in NSG bonemarrow at day 21 when treated with a single dose of (i) wild typeanti-CD117-ADC, (ii) a CK6 variant anti-CD117-ADC with a shorter halflife or (iii) a control. (B) Shows the results of a humanized NSG mousedepletion assay after the administration of a single dose of a wild typeanti-CD117-ADC (mg/kg) in comparison to a CK6 variant anti-CD117-ADCwith a shorter half life (mg/kg) and various controls (i.e., “control,”“isotype-ADC (1 mg/kg)” and “Anti-CD117 (1 mg/kg)”), expressed as theCD34+ cell number per femur (y-axis) as a function of a single injectionof (i) wild type anti-CD117-ADC, (ii) “short half life” variantanti-CD117-ADC or (iii) the controls (x-axis).

FIGS. 8A, 8B and 8C graphically depict the results of an in vivodepletion assay showing that human peripheral lymphocytes are maintained21 days after administration of a single dose of anti-CD117 ADC in ahumanized mouse model. (A) Shows the percentage of human CD33+ cells(relative to baseline) maintained after 21 days in mice treated withvarious concentrations of a single dose of (i) a wild typeanti-CD117-ADC (mg/kg), (ii) a CK6 variant anti-CD117-ADC with a shorterhalf life (mg/kg) or (iii) various controls (i.e., “control,”“isotype-ADC (1 mg/kg)” and “Anti-CD117 (1 mg/kg)”). (B) Shows thepercentage of human CD3+ cells (relative to baseline) maintained after21 days in mice treated with various concentrations of a single dose of(i) a wild type anti-CD117-ADC (mg/kg), (ii) a CK6 variantanti-CD117-ADC with a shorter half life (mg/kg) or (iii) variouscontrols (i.e., “control,” “isotype-ADC (1 mg/kg)” and “Anti-CD117 (1mg/kg)”). (C) Shows the percentage of human CD19+ cells (relative tobaseline) maintained after 21 days in mice treated with variousconcentrations of a single dose of (i) a wild type anti-CD117-ADC(mg/kg), (ii) a CK6 variant anti-CD117-ADC with a shorter half life(mg/kg) or (iii) various controls (i.e., “control,” “isotype-ADC (1mg/kg)” and “Anti-CD117 (1 mg/kg)”).

FIGS. 9A, 9B, 9C and 9D describe measurement of anti-CD117 antibodybinding by Bio-Layer Interferometry (BLI) of the indicated purified IgG(sensor-associated) to purified human CD117 ectodomain (R&D Systems#332-SR) at concentrations of 33.3 nM (top trace) and 11 nM (bottomtrace) as a function of time for the (A) HC-1/LC-1 (Ab1) antibody, (B)the HC-77/LC-77 (Ab 77) antibody, (C) the HC-79/LC-79 (Ab79) antibody,and (D) the HC-81/LC-81 (Ab81) antibody.

FIGS. 10A and 10B demonstrate the measurement of binding by Bio-LayerInterferometry (BLI) of the indicated purified IgG (sensor-associated)to purified human CD117 ectodomain (R&D Systems #332-SR) atconcentrations of 33.3 nM (top traces) and 11 nM (bottom traces) as afunction of time for the (A) HC-85/LC-85 antibody and the (B) HC-1/LC-1antibody.

FIGS. 11A, 11B, 11C, and 11D provide the variable heavy (VH) andvariable light (VL) chain region of the amino acid sequences of CK6,Ab85 and Ab249. (A) Depicts the alignment of the variable heavy (VH)chain regions of CK6 (SEQ ID NO: 161) and Ab85 (SEQ ID NO: 143). (B)Depicts the alignment of the variable light (VL) chain regions of CK6(SEQ ID NO: 162) and Ab85 (SEQ ID NO: 144). (C) Depicts the alignment ofthe variable heavy (VH) chain regions of CK6 (SEQ ID NO: 161) and Ab249(SEQ ID NO: 98). (D) Depicts the alignment of the variable light (VL)chain regions of CK6 (SEQ ID NO: 162) and Ab249 (SEQ ID NO: 102).

FIGS. 12A, 12B and 12C depict the binding by bio-layer interferometry(BLI) of the indicated purified IgG (sensor-associated) to 11 nm (bottomtrace) and 33 nM (top trace) purified human CD117 ectodomain as afunction of time for the (A) CK6 antibody, (B) the HC-85/LC-85 (Ab85)antibody and the (C) HC-249/LC-249 (Ab 249) antibody.

FIGS. 13A and 13B illustrate the fraction of acidic variants present inthe indicated antibody under the indicated incubation conditions(x-axis) for (A) Day 7 (25° C. and 50° C.) and (B) Day 15 (25° C. and50° C.) compared to T₀ as determined by capillary electrophoresis.

FIGS. 14A, 14B, 14C, 14D and 14E depict chromatograms demonstrating theelution profile of (A) the CK6 antibody, (B) the HC-77/LC-77 (Ab77)antibody, (C) the HC-79/LC-79 (Ab79) antibody, (D) the HC-81/LC-81(Ab81) antibody, and (E) the HC-85/LC-85 (Ab85) antibody, under theindication incubation conditions (see legend) after analysis byhydrophobic interaction chromatography (HIC).

FIG. 15 graphically depicts the results of in vitro cell killing assaysthat show Kasumi-1 cell viability as measured in luminescene (RLU) byCelltiter Glo as a function of the indicated anti-CD117 ADC or controlconcentration. The IC50 (M) was determined by non-linear regression4-parameter fit in Graphpad Prism.

FIG. 16 graphically depicts the results of in vitro cell killing assaysthat show viable CD34+ CD90+ cell count as a function of the indicatedanti-CD117 ADC or control concentration. The IC50 (M) was determined bynon-linear regression 4-parameter fit in Graphpad Prism.

FIGS. 17A and 17B graphically depict the results of an in vivo celldepletion assay that shows a 0.3 mg/kg dose of the HC-85/LC-85 antibody(i.e., Ab85) ADC selectively depletes human HSCs in humanized NSG mice.(A) Shows the percent of human myeloid cells present in the peripheralblood of Ab85-ADC or control treated mice, expressed as a percent ofthat cell population prior to treatment (normalized to baseline) isshown for samples collected on day 0, 7, and 14. (B) Shows the absolutenumber of CD34+ cells in the bone marrow of the HC-85/LC-85-ADC (i.e.,Ab85-ADC) or control treated mice 14 days after a single administrationof the ADC or the control (i.e., PBS).

FIG. 18 graphically depicts the results of an in vitro toxicity assayusing human bone marrow CD34+ cells for an anti-CD117 antibody (CK6)site specifically conjugated to various cytotoxic payloads. The resultsshow that the anti-CD117 (CK6) antibody conjugated to amanitin resultedin a >90% depletion of human HSCs in humanized mice at 0.3 mg/kg. Theseresults also show that the calicheamicin and amanitin ADCs demonstratecomparable depletion of HSCs

FIG. 19 graphically depicts hematopoietic stem and progenitor cellnumber in bone marrow. The results show that a single dose of amanitinconjugated anti-CD117 (CD117-AM) eliminates bone marrow HSCs incynomolgus monkeys. Male cynomolgus monkeys received a single i.v. doseof anti-CD117-AM, isotype-AM (IgG1-AM), or unconjugated anti-CD117antibody. Bone marrow HSC counts were determined by flow cytometry 7days post administration. The fraction of HSCs depleted after treatmentwith anti-CD117-AM, IgG1-AM, or unconjugated anti-CD117 was calculatedrelative to the PBS group.

FIG. 20 graphically depicts hematopoietic stem and progenitor cellnumber in bone marrow. The results show that a single dose or afractionated dose of amanitin conjugated anti-CD117 (HC-85-LC 85)modified with a fast half-life Fc (H435A) eliminates bone marrow HSCs incynomolgus monkeys. Male cynomolgus monkeys received a single i.v. dose(0.1 or 0.3 mg/kg) or a fractionated i.v. dose (0.3 mg/kg and 0.2 mg/kgQ3D) of anti-CD117 ADC. Bone marrow HSC counts were determined by flowcytometry 7 days post administration.

FIGS. 21A, 21B, and 21C graphically depict the results of a survivalstudy using three AML-patient-derived xenografts developed fromFLT-3+NPM1+ AML samples showing survival was significantly increased inmouse models administered 0.3 mg/kg anti-CD117-amanitin or 1 mg/kganti-CD117-amanitin. (A) Survival curve of AML-PDX developed fromFLT-3+NPM1+ AML sample J000106132 after single intravenousadministration of ADCs (anti-CD117-AM, isotype-AM), an unconjugatedanti-CD117 antibody or PBS (control). (B) Survival curve of AML-PDXdeveloped from FLT-3+NPM1+ AML sample J000106565 after singleintravenous administration of ADCs (anti-CD117-AM, isotype-AM), anunconjugated anti-CD117 antibody or PBS (control). (C) Survival curve ofAML-PDX developed from FLT-3+NPM1+ AML sample J000106134 after singleintravenous administration of ADCs (anti-CD117-AM, isotype-AM), anunconjugated anti-CD117 antibody or PBS (control).

FIG. 22 demonstrates the measurement of binding by Bio-LayerInterferometry (BLI) of the indicated purified IgG (sensor-associated)to 100 nM purified human CD117 ectodomain (R&D Systems #332-SR) as afunction of time. The purified IgGs correspond to Ab17 (i.e., 017), Ab18(i.e., 018), Ab19 (i.e., 019), Ab20 (i.e., 020), Ab21 (i.e., 021), Ab22(i.e., 022), Ab23 (i.e., 023), Ab24 (i.e., 024), Ab25 (i.e., 025), Ab27(i.e., 027), and Ab28 (i.e., 028).

FIG. 23 demonstrates the measurement of binding by Bio-LayerInterferometry (BLI) of the indicated purified IgG (sensor-associated)to 100 nM purified rhesus CD117 ectodomain as a function of time. Thepurified IgGs correspond to Ab17 (i.e., 017), Ab18 (i.e., 018), Ab19(i.e., 019), Ab20 (i.e., 020), Ab21 (i.e., 021), Ab22 (i.e., 022), Ab23(i.e., 023), Ab24 (i.e., 024), Ab25 (i.e., 025), Ab27 (i.e., 027), andAb28 (i.e., 028).

DETAILED DESCRIPTION

Described herein are isolated anti-CD117 human antibodies that bind tohuman CD117. The antibodies provided herein have many characteristicsmaking them advantageous for therapy, including methods of conditioninghuman patients for stem cell transplantation. For example, antibodiesdisclosed herein, in certain embodiments, have high affinity and a lawoff rate for human CD117, as well as the ability to internalize in cellsexpressing CD117. Further, certain of the antibodies presented hereinhave improved biophysical stability. These features also make theanti-CD117 antibodies disclosed herein advantageous for use inconjugates (antibody drug conjugates (ADCs)) for delivering cytotoxinsto CD117 expressing cells.

The invention provides anti-CD117 antibodies, specifically isolatedhuman anti-CD117 antibodies that bind to the ectodomain of human CD117.The binding regions of the isolated anti-CD117 antibodies identifiedherein are described below and in Table 1, Table 6, and Table 8.

The anti-CD117 antibodies and ADCs described herein can be used inmethods of treating a variety of disorders, such as diseases of a celltype in the hematopoietic lineage, cancers, autoimmune diseases,metabolic disorders, and stem cell disorders, among others. Thecompositions and methods described herein may (i) directly deplete apopulation of cells that give rise to a pathology, such as a populationof cancer cells (e.g., leukemia cells) and autoimmune cells (e.g.,autoreactive T-cells), and/or (ii) deplete a population of endogenoushematopoietic stem cells so as to promote the engraftment oftransplanted hematopoietic stem cells by providing a niche to which thetransplanted cells may home. The foregoing activities can be achieved byadministration of an ADC, antibody, or antigen-binding fragment thereof,capable of binding an antigen expressed by an endogenous disease-causingcell or a hematopoietic stem cell. In the case of direct treatment of adisease, this administration can cause a reduction in the quantity ofthe cells that give rise to the pathology of interest. In the case ofpreparing a patient for hematopoietic stem cell transplant therapy, thisadministration can cause the selective depletion of a population ofendogenous hematopoietic stem cells, thereby creating a vacancy in thehematopoietic tissue, such as the bone marrow, that can subsequently befilled by transplanted, exogenous hematopoietic stem cells. Theinvention is based in part on the discovery that ADCs, antibodies, orantigen-binding fragments thereof, capable of binding CD117 (such asGNNK+ D117) can be administered to a patient to affect both of the aboveactivities. ADCs, antibodies, or antigen-binding fragments thereof, thatbind CD117 can be administered to a patient suffering from a cancer orautoimmune disease to directly deplete a population of cancerous cellsor autoimmune cells, and can also be administered to a patient in needof hematopoietic stem cell transplant therapy in order to promote thesurvival and engraftment potential of transplanted hematopoietic stemcells.

Engraftment of hematopoietic stem cell transplants due to theadministration of anti-CD117 ADCs, antibodies, or antigen-bindingfragments thereof, can manifest in a variety of empirical measurements.For instance, engraftment of transplanted hematopoietic stem cells canbe evaluated by assessing the quantity of competitive repopulating units(CRU) present within the bone marrow of a patient followingadministration of an ADC, antibody or antigen-binding fragment thereofcapable of binding CD117 and subsequent administration of ahematopoietic stem cell transplant. Additionally, one can observeengraftment of a hematopoietic stem cell transplant by incorporating areporter gene, such as an enzyme that catalyzes a chemical reactionyielding a fluorescent, chromophoric, or luminescent product, into avector with which the donor hematopoietic stem cells have beentransfected and subsequently monitoring the corresponding signal in atissue into which the hematopoietic stem cells have homed, such as thebone marrow. One can also observe hematopoietic stem cell engraftment byevaluation of the quantity and survival of hematopoietic stem andprogenitor cells, for instance, as determined by fluorescence activatedcell sorting (FACS) analysis methods known in the art. Engraftment canalso be determined by measuring white blood cell counts in peripheralblood during a post-transplant period, and/or by measuring recovery ofmarrow cells by donor cells in a bone marrow aspirate sample.

The sections that follow provide a description of ADCs, antibodies, orantigen-binding fragments thereof, that can be administered to apatient, such as a patient suffering from a cancer (such as acutemyelogenous leukemia or myelodysplastic syndrome) or autoimmune disease,or a patient in need of hematopoietic stem cell transplant therapy inorder to promote engraftment of hematopoietic stem cell grafts, as wellas methods of administering such therapeutics to a patient (e.g., priorto hematopoietic stem cell transplantation).

Definitions

As used herein, the term “about” refers to a value that is within 10%above or below the value being described. For example, the term “about 5nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the term “amatoxin” refers to a member of the amatoxinfamily of peptides produced by Amanita phalloides mushrooms, or avariant or derivative thereof, such as a variant or derivative thereofcapable of inhibiting RNA polymerase II activity. Amatoxins useful inconjunction with the compositions and methods described herein includecompounds according to, but are not limited to, formula (III), includingα-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, amanullinic acid, or proamanullin. As described herein,amatoxins may be conjugated to an antibody, or antigen-binding fragmentthereof, for instance, by way of a linker moiety (L) (thus forming anADC). Exemplary methods of amatoxin conjugation and linkers useful forsuch processes are described below. Exemplary linker-containingamatoxins useful for conjugation to an antibody, or antigen-bindingfragment, in accordance with the compositions and methods are alsodescribed herein.

Formula (III) is as follows:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

For instance, amatoxins useful in conjunction with the compositions andmethods described herein include compounds according to formula (IIIA),below:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

Amatoxins useful in conjunction with the compositions and methodsdescribed herein also include compounds according to formula (IIIB),below:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

As described herein, amatoxins may be conjugated to an antibody, orantigen-binding fragment thereof, for instance, by way of a linkermoiety (L) (thus forming an ADC). Exemplary methods of amatoxinconjugation and linkers useful for such processes are described below,including Table 3. Exemplary linker-containing amatoxins useful forconjugation to an antibody, or antigen-binding fragment, in accordancewith the compositions and methods described herein are shown instructural formulas (I), (IA), (IB), (II), (IIA), or (IIB), recitedherein.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes monoclonal, genetically engineered, andotherwise modified forms of antibodies, including but not limited tochimeric antibodies, humanized antibodies, heteroconjugate antibodies(e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, andtetrabodies), and antigen binding fragments of antibodies, including,for example, Fab′, F(ab′)₂, Fab, Fv, rIgG, and scFv fragments.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used herein refers to a polypeptide, e.g.,an antibody, that has been identified and separated and/or recoveredfrom a cell or cell culture from which it was expressed. Ordinarily, anisolated antibody will be prepared by at least one purification step.Thus, an “isolated antibody,” refers to an antibody which issubstantially free of other antibodies having different antigenicspecificities. For instance, an isolated antibody that specificallybinds to CD117 is substantially free of antibodies that specificallybind antigens other than CD117.

Unless otherwise indicated, the term “monoclonal antibody” (mAb) ismeant to include both intact molecules, as well as antibody fragments(including, for example, Fab and F(ab′)₂ fragments) that are capable ofspecifically binding to a target protein. As used herein, the Fab andF(ab′)₂ fragments refer to antibody fragments that lack the Fc fragmentof an intact antibody. Examples of these antibody fragments aredescribed herein.

Generally, antibodies comprise heavy and light chains containing antigenbinding regions. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as HCVR or VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH, and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxyl-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

An “intact” or “full length” antibody, as used herein, refers to anantibody having two heavy (H) chain polypeptides and two light (L) chainpolypeptides interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH, and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies can mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system.

The terms “Fc”, “Fc region,” and “Fc domain,” as used herein refer tothe portion of an IgG antibody that correlates to a crystallizablefragment obtained by papain digestion of an IgG molecule. The Fc regioncomprises the C-terminal half of two heavy chains of an IgG moleculethat are linked by disulfide bonds. It has no antigen binding activitybut contains the carbohydrate moiety and binding sites for complementand Fc receptors, including the FcRn receptor. An Fc region contains thesecond constant domain CH2 (e.g., residues at EU positions 231-340 ofIgG1) and the third constant domain CH3 (e.g., residues at EU positions341-447 of human IgG1). As used herein, the Fc region includes the“lower hinge region” (e.g., residues at EU positions 233-239 of IgG1).Fc can refer to this region in isolation, or this region in the contextof an antibody, antibody fragment, or Fc fusion protein. Polymorphismshave been observed at a number of positions in Fc domains, including butnot limited to EU positions 270, 272, 312, 315, 356, and 358, and thusslight differences between the sequences presented in the instantapplication and sequences known in the art can exist. Thus, a “wild typeIgG Fc domain” or “WT IgG Fc domain” refers to any naturally occurringIgG Fc region (i.e., any allele). The sequences of the heavy chains ofhuman IgG1, IgG2, IgG3 and IgG4 can be found in a number of sequencedatabases, for example, at the Uniprot database (www.uniprot.org) underaccession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860(IGHG3_HUMAN), and P01861 (IGHG1_HUMAN), respectively. An example of a“WT” Fc region is provided in SEQ ID NO: 183 (which provides a heavychain constant region containing an Fc region).

The terms “modified Fc region” or “variant Fc region” as used hereinrefers to an IgG Fc domain comprising one or more amino acidsubstitutions, deletions, insertions or modifications introduced at anyposition within the Fc region.

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be, for example, a Fab, F(ab′)₂, scFv, diabody, atriabody, an affibody, a nanobody, an aptamer, or a domain antibody.Examples of binding fragments encompassed of the term “antigen-bindingfragment” of an antibody include, but are not limited to: (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L),and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcontaining two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb including V_(H) and V_(L) domains; (vi) adAb fragment that consists of a V_(H) domain (see, e.g., Ward et al.,Nature 341:544-546, 1989); (vii) a dAb which consists of a V_(H) or aV_(L) domain; (viii) an isolated complementarity determining region(CDR); and (ix) a combination of two or more (e.g., two, three, four,five, or six) isolated CDRs which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); see, forexample, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments canbe obtained using conventional techniques known to those of skill in theart, and the fragments can be screened for utility in the same manner asintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, enzymatic or chemical cleavage of intactimmunoglobulins, or, in certain cases, by chemical peptide synthesisprocedures known in the art.

As used herein, the term “anti-CD117 antibody” or “an antibody thatbinds to CD117” refers to an antibody that is capable of binding CD117with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting CD117.

As used herein, the term “bispecific antibody” refers to, for example, amonoclonal, often a human or humanized antibody that is capable ofbinding at least two different antigens. For instance, one of thebinding specificities can be directed towards a hematopoietic stem cellsurface antigen, CD117 (e.g., GNNK+ CD117), and the other canspecifically bind a different hematopoietic stem cell surface antigen oranother cell surface protein, such as a receptor or receptor subunitinvolved in a signal transduction pathway that potentiates cell growth,among others.

As used herein, the term “complementarity determining region” (CDR)refers to a hypervariable region found both in the light chain and theheavy chain variable domains of an antibody. The more highly conservedportions of variable domains are referred to as framework regions (FRs).The amino acid positions that delineate a hypervariable region of anantibody can vary, depending on the context and the various definitionsknown in the art. Some positions within a variable domain may be viewedas hybrid hypervariable positions in that these positions can be deemedto be within a hypervariable region under one set of criteria whilebeing deemed to be outside a hypervariable region under a different setof criteria. One or more of these positions can also be found inextended hypervariable regions. The antibodies described herein maycontain modifications in these hybrid hypervariable positions. Thevariable domains of native heavy and light chains each contain fourframework regions that primarily adopt a β-sheet configuration,connected by three CDRs, which form loops that connect, and in somecases form part of, the β-sheet structure. The CDRs in each chain areheld together in close proximity by the framework regions in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the otherantibody chains, contribute to the formation of the target binding siteof antibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, National Institute of Health, Bethesda, Md., 1987). In certainembodiments, numbering of immunoglobulin amino acid residues isperformed according to the immunoglobulin amino acid residue numberingsystem of Kabat et al., unless otherwise indicated (although anyantibody numbering scheme, including, but not limited to IMGT andChothia, can be utilized).

As used herein, the terms “condition” and “conditioning” refer toprocesses by which a patient is prepared for receipt of a transplantcontaining hematopoietic stem cells. Such procedures promote theengraftment of a hematopoietic stem cell transplant (for instance, asinferred from a sustained increase in the quantity of viablehematopoietic stem cells within a blood sample isolated from a patientfollowing a conditioning procedure and subsequent hematopoietic stemcell transplantation. According to the methods described herein, apatient may be conditioned for hematopoietic stem cell transplanttherapy by administration to the patient of an ADC, an antibody orantigen-binding fragment thereof capable of binding an antigen expressedby hematopoietic stem cells, such as CD117 (e.g., GNNK+ CD117). Asdescribed herein, the antibody may be covalently conjugated to acytotoxin so as to form an ADC. Administration of an antibody,antigen-binding fragment thereof, or ADC capable of binding one or moreof the foregoing antigens to a patient in need of hematopoietic stemcell transplant therapy can promote the engraftment of a hematopoieticstem cell graft, for example, by selectively depleting endogenoushematopoietic stem cells, thereby creating a vacancy filled by anexogenous hematopoietic stem cell transplant.

As used herein, the term “conjugate” refers to a compound formed by thechemical bonding of a reactive functional group of one molecule, such asan antibody or antigen-binding fragment thereof, with an appropriatelyreactive functional group of another molecule, such as a cytotoxindescribed herein. Conjugates may include a linker between the twomolecules bound to one another. Examples of linkers that can be used forthe formation of a conjugate include peptide-containing linkers, such asthose that contain naturally occurring or non-naturally occurring aminoacids, such as D-amino acids. Linkers can be prepared using a variety ofstrategies described herein and known in the art. Depending on thereactive components therein, a linker may be cleaved, for example, byenzymatic hydrolysis, photolysis, hydrolysis under acidic conditions,hydrolysis under basic conditions, oxidation, disulfide reduction,nucleophilic cleavage, or organometallic cleavage (see, for example,Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012). The foregoingconjugates are also referred to interchangeably herein as a “drugantibody conjugate”, an “antibody drug conjugate” and an “ADC”.

As used herein, the term “coupling reaction” refers to a chemicalreaction in which two or more substituents suitable for reaction withone another react so as to form a chemical moiety that joins (e.g.,covalently) the molecular fragments bound to each substituent. Couplingreactions include those in which a reactive substituent bound to afragment that is a cytotoxin, such as a cytotoxin known in the art ordescribed herein, reacts with a suitably reactive substituent bound to afragment that is an antibody, or antigen-binding fragment thereof, suchas an antibody, or antigen-binding fragment thereof, specific for CD117(such as GNNK+ CD117) known in the art or described herein. Examples ofsuitably reactive substituents include a nucleophile/electrophile pair(e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or athiol/α,β-unsaturated carbonyl pair, among others), a diene/dienophilepair (e.g., an azide/alkyne pair, among others), and the like. Couplingreactions include, without limitation, thiol alkylation, hydroxylalkylation, amine alkylation, amine condensation, amidation,esterification, disulfide formation, cycloaddition (e.g., [4+2]Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others),nucleophilic aromatic substitution, electrophilic aromatic substitution,and other reactive modalities known in the art or described herein.

As used herein, “CRU (competitive repopulating unit)” refers to a unitof measure of long-term engrafting stem cells, which can be detectedafter in-vivo transplantation.

As used herein, “drug-to-antibody ratio” or “DAR” refers to the numberof cytotoxins, e.g., amatoxin, attached to the antibody of an ADC. TheDAR of an ADC can range from 1 to 8, although higher loads are alsopossible depending on the number of linkage sites on an antibody. Thus,in certain embodiments, an ADC described herein has a DAR of 1, 2, 3, 4,5, 6, 7, or 8.

As used herein, the term “donor” refers to a human or animal from whichone or more cells are isolated prior to administration of the cells, orprogeny thereof, into a recipient. The one or more cells may be, forexample, a population of hematopoietic stem cells.

As used herein, the term “diabody” refers to a bivalent antibodycontaining two polypeptide chains, in which each polypeptide chainincludes V_(H) and V_(L) domains joined by a linker that is too short(e.g., a linker composed of five amino acids) to allow forintramolecular association of V_(H) and V_(L) domains on the samepeptide chain. This configuration forces each domain to pair with acomplementary domain on another polypeptide chain so as to form ahomodimeric structure. Accordingly, the term “triabody” refers totrivalent antibodies containing three peptide chains, each of whichcontains one V_(H) domain and one V_(L) domain joined by a linker thatis exceedingly short (e.g., a linker composed of 1-2 amino acids) topermit intramolecular association of V_(H) and V_(L) domains within thesame peptide chain. In order to fold into their native structures,peptides configured in this way typically trimerize so as to positionthe V_(H) and V_(L) domains of neighboring peptide chains spatiallyproximal to one another (see, for example, Holliger et al., Proc. Natl.Acad. Sci. USA 90:6444-48, 1993).

As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”)refers to an antibody that combines the target-binding variable domainsof two monoclonal antibodies via linkers to create a tetravalent,dual-targeting single agent (see, for example, Gu et al., Meth.Enzymol., 502:25-41, 2012).

As used herein, the term “endogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T-lymphocyte, or B-lymphocyte) that is found naturally in aparticular organism, such as a human patient.

As used herein, the term “engraftment potential” is used to refer to theability of hematopoietic stem and progenitor cells to repopulate atissue, whether such cells are naturally circulating or are provided bytransplantation. The term encompasses all events surrounding or leadingup to engraftment, such as tissue homing of cells and colonization ofcells within the tissue of interest. The engraftment efficiency or rateof engraftment can be evaluated or quantified using any clinicallyacceptable parameter as known to those of skill in the art and caninclude, for example, assessment of competitive repopulating units(CRU); incorporation or expression of a marker in tissue(s) into whichstem cells have homed, colonized, or become engrafted; or by evaluationof the progress of a subject through disease progression, survival ofhematopoietic stem and progenitor cells, or survival of a recipient.Engraftment can also be determined by measuring white blood cell countsin peripheral blood during a post-transplant period. Engraftment canalso be assessed by measuring recovery of marrow cells by donor cells ina bone marrow aspirate sample.

As used herein, the term “exogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T-lymphocyte, or B-lymphocyte) that is not found naturally in aparticular organism, such as a human patient. Exogenous substancesinclude those that are provided from an external source to an organismor to cultured matter extracted therefrom.

As used herein, the term “framework region” or “FW region” includesamino acid residues that are adjacent to the CDRs of an antibody orantigen-binding fragment thereof. FW region residues may be present in,for example, human antibodies, humanized antibodies, monoclonalantibodies, antibody fragments, Fab fragments, single chain antibodyfragments, scFv fragments, antibody domains, and bispecific antibodies,among others.

As used herein, the term “hematopoietic stem cells” (“HSCs”) refers toimmature blood cells having the capacity to self-renew and todifferentiate into mature blood cells containing diverse lineagesincluding but not limited to granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B-cells and T-cells). Such cells may include CD34⁺ cells. CD34⁺cells are immature cells that express the CD34 cell surface marker. Inhumans, CD34+ cells are believed to include a subpopulation of cellswith the stem cell properties defined above, whereas in mice, HSCs areCD34−. In addition, HSCs also refer to long term repopulating HSCs(LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and ST-HSCsare differentiated, based on functional potential and on cell surfacemarker expression. For example, human HSCs are CD34+, CD38−, CD45RA−,CD90+, CD49F+, and lin− (negative for mature lineage markers includingCD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). Inmice, bone marrow LT-HSCs are CD34−, SCA-1+, C-kit+, CD135−,Slamfl/CD150+, CD48−, and lin− (negative for mature lineage markersincluding Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereasST-HSCs are CD34+, SCA-1+, C-kit+, CD135−, Slamfl/CD150+, and lin−(negative for mature lineage markers including Ter119, CD11b, Gr1, CD3,CD4, CD8, B220, IL7ra). In addition, ST-HSCs are less quiescent and moreproliferative than LT-HSCs under homeostatic conditions. However, LT-HSChave greater self renewal potential (i.e., they survive throughoutadulthood, and can be serially transplanted through successiverecipients), whereas ST-HSCs have limited self renewal (i.e., theysurvive for only a limited period of time, and do not possess serialtransplantation potential). Any of these HSCs can be used in the methodsdescribed herein. ST-HSCs are particularly useful because they arehighly proliferative and thus, can more quickly give rise todifferentiated progeny.

As used herein, the term “hematopoietic stem cell functional potential”refers to the functional properties of hematopoietic stem cells whichinclude 1) multi-potency (which refers to the ability to differentiateinto multiple different blood lineages including, but not limited to,granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets),monocytes (e.g., monocytes, macrophages), dendritic cells, microglia,osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2)self-renewal (which refers to the ability of hematopoietic stem cells togive rise to daughter cells that have equivalent potential as the mothercell, and further that this ability can repeatedly occur throughout thelifetime of an individual without exhaustion), and 3) the ability ofhematopoietic stem cells or progeny thereof to be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis.

As used herein, the term “human antibody” is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. A human antibody may include aminoacid residues not encoded by human germline immunoglobulin sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or during gene rearrangement or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. A human antibody can be produced in a human cell(for example, by recombinant expression) or by a non-human animal or aprokaryotic or eukaryotic cell that is capable of expressingfunctionally rearranged human immunoglobulin (such as heavy chain and/orlight chain) genes. When a human antibody is a single chain antibody, itcan include a linker peptide that is not found in native humanantibodies. For example, an Fv can contain a linker peptide, such as twoto about eight glycine or other amino acid residues, which connects thevariable region of the heavy chain and the variable region of the lightchain. Such linker peptides are considered to be of human origin. Humanantibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences. Human antibodies can also be producedusing transgenic mice that are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes (see, for example, PCT Publication Nos. WO 1998/24893; WO1992/01047; WO 1996/34096; WO 1996/33735; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598).

As used herein, patients that are “in need of” a hematopoietic stem celltransplant include patients that exhibit a defect or deficiency in oneor more blood cell types, as well as patients having a stem celldisorder, autoimmune disease, cancer, or other pathology describedherein. Hematopoietic stem cells generally exhibit 1) multi-potency, andcan thus differentiate into multiple different blood lineages including,but not limited to, granulocytes (e.g., promyelocytes, neutrophils,eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B-cells and T-cells), 2) self-renewal, and can thus give rise todaughter cells that have equivalent potential as the mother cell, and 3)the ability to be reintroduced into a transplant recipient whereuponthey home to the hematopoietic stem cell niche and re-establishproductive and sustained hematopoiesis. Hematopoietic stem cells canthus be administered to a patient defective or deficient in one or morecell types of the hematopoietic lineage in order to re-constitute thedefective or deficient population of cells in vivo. For example, thepatient may be suffering from cancer, and the deficiency may be causedby administration of a chemotherapeutic agent or other medicament thatdepletes, either selectively or non-specifically, the cancerous cellpopulation. Additionally or alternatively, the patient may be sufferingfrom a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), suchas sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, andWiskott-Aldrich syndrome. The subject may be one that is suffering fromadenosine deaminase severe combined immunodeficiency (ADA SCID),HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, andSchwachman-Diamond syndrome. The subject may have or be affected by aninherited blood disorder (e.g., sickle cell anemia) or an autoimmunedisorder. Additionally or alternatively, the subject may have or beaffected by a malignancy, such as neuroblastoma or a hematologic cancer.For instance, the subject may have a leukemia, lymphoma, or myeloma. Insome embodiments, the subject has acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiplemyeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Insome embodiments, the subject has myelodysplastic syndrome. In someembodiments, the subject has an autoimmune disease, such as scleroderma,multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1diabetes, or another autoimmune pathology described herein. In someembodiments, the subject is in need of chimeric antigen receptor T-cell(CART) therapy. In some embodiments, the subject has or is otherwiseaffected by a metabolic storage disorder. The subject may suffer orotherwise be affected by a metabolic disorder selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, metachromaticleukodystrophy, or any other diseases or disorders which may benefitfrom the treatments and therapies disclosed herein and including,without limitation, severe combined immunodeficiency, Wiscott-Aldrichsyndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, sickle cell disease,systemic sclerosis, systemic lupus erythematosus, multiple sclerosis,juvenile rheumatoid arthritis and those diseases, or disorders describedin “Bone Marrow Transplantation for Non-Malignant Disease,” ASHEducation Book, 1:319-338 (2000), the disclosure of which isincorporated herein by reference in its entirety as it pertains topathologies that may be treated by administration of hematopoietic stemcell transplant therapy. Additionally or alternatively, a patient “inneed of” a hematopoietic stem cell transplant may one that is or is notsuffering from one of the foregoing pathologies, but nonethelessexhibits a reduced level (e.g., as compared to that of an otherwisehealthy subject) of one or more endogenous cell types within thehematopoietic lineage, such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes. One of skill in the art canreadily determine whether one's level of one or more of the foregoingcell types, or other blood cell type, is reduced with respect to anotherwise healthy subject, for instance, by way of flow cytometry andfluorescence activated cell sorting (FACS) methods, among otherprocedures, known in the art.

As used herein, the term “recipient” refers to a patient that receives atransplant, such as a transplant containing a population ofhematopoietic stem cells. The transplanted cells administered to arecipient may be, e.g., autologous, syngeneic, or allogeneic cells.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, and cells) taken from a subject.

As used herein, the term “scFv” refers to a single chain Fv antibody inwhich the variable domains of the heavy chain and the light chain froman antibody have been joined to form one chain. scFv fragments contain asingle polypeptide chain that includes the variable region of anantibody light chain (V_(L)) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) andthe variable region of an antibody heavy chain (V_(H)) (e.g., CDR-H1,CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins theV_(L) and V_(H) regions of a scFv fragment can be a peptide linkercomposed of proteinogenic amino acids. Alternative linkers can be usedto so as to increase the resistance of the scFv fragment to proteolyticdegradation (for example, linkers containing D-amino acids), in order toenhance the solubility of the scFv fragment (for example, hydrophiliclinkers such as polyethylene glycol-containing linkers or polypeptidescontaining repeating glycine and serine residues), to improve thebiophysical stability of the molecule (for example, a linker containingcysteine residues that form intramolecular or intermolecular disulfidebonds), or to attenuate the immunogenicity of the scFv fragment (forexample, linkers containing glycosylation sites). It will also beunderstood by one of ordinary skill in the art that the variable regionsof the scFv molecules described herein can be modified such that theyvary in amino acid sequence from the antibody molecule from which theywere derived. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at amino acid residuescan be made (e.g., in CDR and/or framework residues) so as to preserveor enhance the ability of the scFv to bind to the antigen recognized bythe corresponding antibody.

As used herein, the terms “subject” and “patient” refer to an organism,such as a human, that receives treatment for a particular disease orcondition as described herein. For instance, a patient, such as a humanpatient, may receive treatment prior to hematopoietic stem celltransplant therapy in order to promote the engraftment of exogenoushematopoietic stem cells.

As used herein, the phrase “substantially cleared from the blood” refersto a point in time following administration of a therapeutic agent (suchas an anti-CD117 antibody, or antigen-binding fragment thereof) to apatient when the concentration of the therapeutic agent in a bloodsample isolated from the patient is such that the therapeutic agent isnot detectable by conventional means (for instance, such that thetherapeutic agent is not detectable above the noise threshold of thedevice or assay used to detect the therapeutic agent). A variety oftechniques known in the art can be used to detect antibodies, antibodyfragments, and protein ligands, such as ELISA-based detection assaysknown in the art or described herein. Additional assays that can be usedto detect antibodies, or antibody fragments, include immunoprecipitationtechniques and immunoblot assays, among others known in the art.

As used herein, the phrase “stem cell disorder” broadly refers to anydisease, disorder, or condition that may be treated or cured byconditioning a subject's target tissues, and/or by ablating anendogenous stem cell population in a target tissue (e.g., ablating anendogenous hematopoietic stem or progenitor cell population from asubject's bone marrow tissue) and/or by engrafting or transplanting stemcells in a subject's target tissues. For example, Type I diabetes hasbeen shown to be cured by hematopoietic stem cell transplant and maybenefit from conditioning in accordance with the compositions andmethods described herein. Additional disorders that can be treated usingthe compositions and methods described herein include, withoutlimitation, sickle cell anemia, thalassemias, Fanconi anemia, aplasticanemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromaticleukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamondsyndrome. Additional diseases that may be treated using the patientconditioning and/or hematopoietic stem cell transplant methods describedherein include inherited blood disorders (e.g., sickle cell anemia) andautoimmune disorders, such as scleroderma, multiple sclerosis,ulcerative colitis, and Crohn's disease. Additional diseases that may betreated using the conditioning and/or transplantation methods describedherein include a malignancy, such as a neuroblastoma or a hematologiccancer, such as leukemia, lymphoma, and myeloma. For instance, thecancer may be acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuselarge B-cell lymphoma, or non-Hodgkin's lymphoma. Additional diseasestreatable using the conditioning and/or transplantation methodsdescribed herein include myelodysplastic syndrome. In some embodiments,the subject has or is otherwise affected by a metabolic storagedisorder. For example, the subject may suffer or otherwise be affectedby a metabolic disorder selected from the group consisting of glycogenstorage diseases, mucopolysaccharidoses, Gaucher's Disease, HurlersDisease, sphingolipidoses, metachromatic leukodystrophy, or any otherdiseases or disorders which may benefit from the treatments andtherapies disclosed herein and including, without limitation, severecombined immunodeficiency, Wiscott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditarylymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storagediseases, thalassemia major, sickle cell disease, systemic sclerosis,systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoidarthritis and those diseases, or disorders described in “Bone MarrowTransplantation for Non-Malignant Disease,” ASH Education Book,1:319-338 (2000), the disclosure of which is incorporated herein byreference in its entirety as it pertains to pathologies that may betreated by administration of hematopoietic stem cell transplant therapy.

As used herein, the term “transfection” refers to any of a wide varietyof techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, such as electroporation,lipofection, calcium-phosphate precipitation, DEAE-dextran transfectionand the like.

As used herein, the terms “treat” or “treatment” refers to reducing theseverity and/or frequency of disease symptoms, eliminating diseasesymptoms and/or the underlying cause of said symptoms, reducing thefrequency or likelihood of disease symptoms and/or their underlyingcause, and improving or remediating damage caused, directly orindirectly, by disease. Beneficial or desired clinical results include,but are not limited to, promoting the engraftment of exogenoushematopoietic cells in a patient following antibody conditioning therapyas described herein and subsequent hematopoietic stem cell transplanttherapy. Additional beneficial results include an increase in the cellcount or relative concentration of hematopoietic stem cells in a patientin need of a hematopoietic stem cell transplant following conditioningtherapy and subsequent administration of an exogenous hematopoietic stemcell graft to the patient. Beneficial results of therapy describedherein may also include an increase in the cell count or relativeconcentration of one or more cells of hematopoietic lineage, such as amegakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast,basophil, neutrophil, eosinophil, microglial cell, granulocyte,monocyte, osteoclast, antigen-presenting cell, macrophage, dendriticcell, natural killer cell, T-lymphocyte, or B-lymphocyte, followingconditioning therapy and subsequent hematopoietic stem cell transplanttherapy. Additional beneficial results may include the reduction inquantity of a disease-causing cell population, such as a population ofcancer cells (e.g., CD117+ leukemic cells) or autoimmune cells (e.g.,CD117+ autoimmune lymphocytes, such as a CD117+ T-cell that expresses aT-cell receptor that cross-reacts with a self antigen). Insofar as themethods of the present invention are directed to preventing disorders,it is understood that the term “prevent” does not require that thedisease state be completely thwarted. Rather, as used herein, the termpreventing refers to the ability of the skilled artisan to identify apopulation that is susceptible to disorders, such that administration ofthe compounds of the present invention may occur prior to onset of adisease. The term does not imply that the disease state is completelyavoided.

As used herein, the terms “variant” and “derivative” are usedinterchangeably and refer to naturally-occurring, synthetic, andsemi-synthetic analogues of a compound, peptide, protein, or othersubstance described herein. A variant or derivative of a compound,peptide, protein, or other substance described herein may retain orimprove upon the biological activity of the original material.

As used herein, the term “vector” includes a nucleic acid vector, suchas a plasmid, a DNA vector, a plasmid, a RNA vector, virus, or othersuitable replicon. Expression vectors described herein may contain apolynucleotide sequence as well as, for example, additional sequenceelements used for the expression of proteins and/or the integration ofthese polynucleotide sequences into the genome of a mammalian cell.Certain vectors that can be used for the expression of antibodies andantibody fragments of the invention include plasmids that containregulatory sequences, such as promoter and enhancer regions, whichdirect gene transcription. Other useful vectors for expression ofantibodies and antibody fragments contain polynucleotide sequences thatenhance the rate of translation of these genes or improve the stabilityor nuclear export of the mRNA that results from gene transcription.These sequence elements may include, for example, 5′ and 3′ untranslatedregions and a polyadenylation signal site in order to direct efficienttranscription of the gene carried on the expression vector. Theexpression vectors described herein may also contain a polynucleotideencoding a marker for selection of cells that contain such a vector.Examples of a suitable marker include genes that encode resistance toantibiotics, such as ampicillin, chloramphenicol, kanamycin, andnourseothricin.

The term “acyl” as used herein refers to —C(═O)R, wherein R is hydrogen(“aldehyde”), alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heteroaryl, orheterocyclyl, as defined herein. Non-limiting examples include formyl,acetyl, propanoyl, benzoyl, and acryloyl.

As used herein, the term “alkyl” refers to a straight- or branched-chainalkyl group having, for example, from 1 to 20 carbon atoms in the chain.Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl,hexyl, isohexyl, and the like.

As used herein, the term “alkylene” refers to a straight- orbranched-chain divalent alkyl group. The divalent positions may be onthe same or different atoms within the alkyl chain. Examples of alkyleneinclude methylene, ethylene, propylene, isopropylene, and the like.

As used herein, the term “heteroalkyl” refers to a straight orbranched-chain alkyl group having, for example, from 1 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkylene” refers to a straight- orbranched-chain divalent heteroalkyl group. The divalent positions may beon the same or different atoms within the heteroalkyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “alkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkenyl groups include vinyl, propenyl,isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.

As used herein, the term “alkenylene” refers to a straight- orbranched-chain divalent alkenyl group. The divalent positions may be onthe same or different atoms within the alkenyl chain. Examples ofalkenylene include ethenylene, propenylene, isopropenylene, butenylene,and the like.

As used herein, the term “heteroalkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkenylene” refers to a straight- orbranched-chain divalent heteroalkenyl group. The divalent positions maybe on the same or different atoms within the heteroalkenyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “alkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkynyl groups include propargyl,butynyl, pentynyl, hexynyl, and the like.

As used herein, the term “alkynylene” refers to a straight- orbranched-chain divalent alkynyl group. The divalent positions may be onthe same or different atoms within the alkynyl chain.

As used herein, the term “heteroalkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkynylene” refers to a straight- orbranched-chain divalent heteroalkynyl group. The divalent positions maybe on the same or different atoms within the heteroalkynyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “cycloalkyl” refers to a monocyclic, or fused,bridged, or spiro polycyclic ring structure that is saturated and has,for example, from 3 to 12 carbon ring atoms. Examples of cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.

As used herein, the term “cycloalkylene” refers to a divalent cycloalkylgroup. The divalent positions may be on the same or different atomswithin the ring structure. Examples of cycloalkylene includecyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and thelike.

As used herein, the term “heterocyloalkyl” refers to a monocyclic, orfused, bridged, or spiro polycyclic ring structure that is saturated andhas, for example, from 3 to 12 ring atoms per ring structure selectedfrom carbon atoms and heteroatoms selected from, e.g., nitrogen, oxygen,and sulfur, among others. The ring structure may contain, for example,one or more oxo groups on carbon, nitrogen, or sulfur ring members.Examples of heterocycloalkyls include by way of example and notlimitation dihydroypyridyl, tetrahydropyridyl (piperidyl),tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl,and morpholinyl.

As used herein, the term “heterocycloalkylene” refers to a divalentheterocyclolalkyl group. The divalent positions may be on the same ordifferent atoms within the ring structure.

As used herein, the term “aryl” refers to a monocyclic or multicyclicaromatic ring system containing, for example, from 6 to 19 carbon atoms.Aryl groups include, but are not limited to, phenyl, fluorenyl,naphthyl, and the like. The divalent positions may be one or moreheteroatoms.

As used herein, the term “arylene” refers to a divalent aryl group. Thedivalent positions may be on the same or different atoms.

As used herein, the term “heteroaryl” refers to a monocyclicheteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromaticgroup in which one or more ring atoms is a heteroatom, e.g., nitrogen,oxygen, or sulfur. Heteroaryl groups include pyridyl, pyrrolyl, furyl,thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl,isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl,isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl,benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, pthalazinyl,quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl,pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl,tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl,purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, and the like.

As used herein, the term “heteroarylene” refers to a divalent heteroarylgroup. The divalent positions may be on the same or different atoms. Thedivalent positions may be one or more heteroatoms.

Unless otherwise constrained by the definition of the individualsubstituent, the foregoing chemical moieties, such as “alkyl”,“alkylene”, “heteroalkyl”, “heteroalkylene”, “alkenyl”, “alkenylene”,“heteroalkenyl”, “heteroalkenylene”, “alkynyl”, “alkynylene”,“heteroalkynyl”, “heteroalkynylene”, “cycloalkyl”, “cycloalkylene”,“heterocyclolalkyl”, heterocycloalkylene”, “aryl,” “arylene”,“heteroaryl”, and “heteroarylene” groups can optionally be substitutedwith, for example, from 1 to 5 substituents selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl,amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl,alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl,alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy,mercapto, nitro, and the like. Typical substituents include, but are notlimited to, —X, —R, —OH, —OR, —SH, —SR, NH₂, —NHR, —N(R)₂, —N⁺(R)₃,—CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, —N₃, —NC(═O)H, —NC(═O)R,—C(═O)H, —C(═O)R, —C(═O)NH₂, —C(═O)N(R)₂, —SO₃—, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NH₂, —S(═O)₂N(R)₂, —S(═O)R, —OP(═O)(OH)₂,—OP(═O)(OR)₂, —P(═O)(OR)₂, —PO₃, —PO₃H₂, —C(═O)X, —C(═S)R, —CO₂H, —CO₂R,—CO₂—, —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NH₂, —C(═O)N(R)₂, —C(═S)NH₂,—C(═S)N(R)₂, —C(═NH)NH₂, and —C(═NR)N(R)₂; wherein each X isindependently selected for each occasion from F, Cl, Br, and I; and eachR is independently selected for each occasion from alkyl, aryl,heterocycloalkyl or heteroaryl, protecting group and prodrug moiety.Wherever a group is described as “optionally substituted,” that groupcan be substituted with one or more of the above substituents,independently for each occasion. The substitution may include situationsin which neighboring substituents have undergone ring closure, such asring closure of vicinal functional substituents, to form, for instance,lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals,aminals, and hemiaminals, formed by ring closure, for example, tofurnish a protecting group.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene,”“alkenylene,” “arylene,” “heterocycloalkylene,” and the like.

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated

Anti-CD117 Antibodies

The present invention is based in part on the discovery of novelanti-CD117 antibodies and antigen binding portions thereof that areuseful for therapeutic purposes. The present invention is also based inpart on the discovery that antibodies, or antigen-binding fragmentsthereof, capable of binding CD117, such as GNNK+ CD117, can be used astherapeutic agents alone or as ADCs to (i) treat cancers (such as acutemyelogenous leukemia or myelodysplastic syndrome) and autoimmunediseases characterized by CD117+ cells and (ii) promote the engraftmentof transplanted hematopoietic stem cells in a patient in need oftransplant therapy. These therapeutic activities can be caused, forinstance, by the binding of anti-CD117 antibodies, or antigen-bindingfragments thereof, to CD117 (e.g., GNNK+ CD117) expressed on the surfaceof a cell, such as a cancer cell, autoimmune cell, or hematopoietic stemcell and subsequently inducing cell death. The depletion of endogenoushematopoietic stem cells can provide a niche toward which transplantedhematopoietic stem cells can home, and subsequently establish productivehematopoiesis. In this way, transplanted hematopoietic stem cells maysuccessfully engraft in a patient, such as human patient suffering froma stem cell disorder described herein.

Antibodies and antigen-binding fragments capable of binding human CD117(also referred to as c-Kit, mRNA NCBI Reference Sequence: NM_000222.2,Protein NCBI Reference Sequence: NP_000213.1), including those capableof binding GNNK+ CD117, can be used in conjunction with the compositionsand methods described herein in order to condition a patient forhematopoietic stem cell transplant therapy. Polymorphisms affecting thecoding region or extracellular domain of CD117 in a significantpercentage of the population are not currently well-known innon-oncology indications. There are at least four isoforms of CD117 thathave been identified, with the potential of additional isoformsexpressed in tumor cells. Two of the CD117 isoforms are located on theintracellular domain of the protein, and two are present in the externaljuxtamembrane region. The two extracellular isoforms, GNNK+ and GNNK−,differ in the presence (GNNK+) or absence (GNNK−) of a 4 amino acidsequence. These isoforms are reported to have the same affinity for theligand (SCF), but ligand binding to the GNNK− isoform was reported toincrease internalization and degradation. The GNNK+ isoform can be usedas an immunogen in order to generate antibodies capable of bindingCD117, as antibodies generated against this isoform will be inclusive ofthe GNNK+ and GNNK− proteins.

The disclosure provides novel anti-CD117 antibodies whose heavy andlight chain amino acid sequences are provided in Table 1, Table 6, Table8, and Table 16. Thus, included in the disclosure is anti-CD117 antibodydrug conjugates comprising binding regions (heavy and light chain CDRsor variable regions) as set forth in SEQ ID Nos: 7 to 168. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 8. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 10. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 11. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:12. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 13. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 14. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 15. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 16. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 17. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:18. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 19. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 20. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 21. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 22. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 23. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 24, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:25. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 26, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 27. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 28, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 29. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 30, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 31. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:32, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 33. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 34, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO:35. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 36, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:37. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 38, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 39. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 40, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 41. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 32, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 42. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:43, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 44. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 45, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 46. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 47, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:48. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 49, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 50. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 51, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 52. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 53, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 54. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:55, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 56. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 57, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 58. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 9, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:60. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 61, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 50. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 62, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 63. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 64, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 65. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:66, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 67. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 68, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 69. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 70, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:71. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 72, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 73. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 74, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 75. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 76, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 77. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:78, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 79. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 80, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 81. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 82, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:83. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 84, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 85. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 86, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 87. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 88. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 89. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 7, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 90. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 7, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:91. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 7, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 92. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 94. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO:95. In one embodiment, the anti-CD117 antibody, orantigen binding portion thereof, comprises a heavy chain variable regionas set forth in the amino acid sequence of SEQ ID NO: 7, and a lightchain variable region as set forth in the amino acid sequence of SEQ IDNO: 96.

In one embodiment, the anti-CD117 antibody, or antigen binding portionthereof, comprises a heavy chain variable region as set forth in theamino acid sequence of SEQ ID NO: 7, and a light chain variable regionas set forth in the amino acid sequence of SEQ ID NO: 97. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 7, and a light chain variable region as set forthin the amino acid sequence of SEQ ID NO: 97. In one embodiment, theanti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 143, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 144. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO:151, and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 152. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 143, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 156. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 158, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:156. In one embodiment, the anti-CD117 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region as set forth inthe amino acid sequence of SEQ ID NO: 160, and a light chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 152. In oneembodiment, the anti-CD117 antibody, or antigen binding portion thereof,comprises a heavy chain variable region as set forth in the amino acidsequence of SEQ ID NO: 98, and a light chain variable region as setforth in the amino acid sequence of SEQ ID NO: 99. In one embodiment,the anti-CD117 antibody, or antigen binding portion thereof, comprises aheavy chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 7, and a light chain variable region as set forth in theamino acid sequence of SEQ ID NO: 99. In one embodiment, the anti-CD117antibody, or antigen binding portion thereof, comprises a heavy chainvariable region as set forth in the amino acid sequence of SEQ ID NO: 7,and a light chain variable region as set forth in the amino acidsequence of SEQ ID NO: 100. In one embodiment, the anti-CD117 antibody,or antigen binding portion thereof, comprises a heavy chain variableregion as set forth in the amino acid sequence of SEQ ID NO: 98, and alight chain variable region as set forth in the amino acid sequence ofSEQ ID NO: 101. In one embodiment, the anti-CD117 antibody, or antigenbinding portion thereof, comprises a heavy chain variable region as setforth in the amino acid sequence of SEQ ID NO: 98, and a light chainvariable region as set forth in the amino acid sequence of SEQ ID NO:102.

In one embodiment, the antibody is an intact antibody comprising a heavychain and a light chain variable region as set forth in Table 1. In oneembodiment, the anti-CD117 antibody is engineered to have a short halflife.

TABLE 1 Antibody heavy and light chain variable region amino acidsequences Antibody Type of name Chain Amino acid sequence HC-1 hIgG1QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-1 hKappaAIQLTQSPSSLSASVGDRVTITCRASQGVSSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 8) HC-2 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-2 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGIRTDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 9) HC-3 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-3 hKappaAIRMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPGKTPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 10) HC-4 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-4 hKappaAIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVDIK (SEQ IDNO: 11) HC-5 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-5 hKappaNIQMTQSPSSLSASVGDRVTITCRASQAISDYLAWFQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNSYPLTFGGGTKVEIK (SEQ IDNO: 12) HC-6 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-6 hKappaAIRMTQSPSSLSASVGDRVIIACRASQGIGGALAWYQQKPGNAPKVLVYDASTLESGVPSRFSGGGSGTDFTLTISSLQPEDFATYYC QQFNSYPLTFGGGTKLEIK (SEQ IDNO: 13) HC-7 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-7 hKappaDIAMTQSPPSLSAFVGDRVTITCRASQGIISSLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTIRSLQPEDFATYYCQQ FNSYPLTFGGGTKLEIK (SEQ IDNO: 14) HC-8 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-8 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKAGKAPKVLISDASSLESGVPSRFSGSGSGTDFTLSISSLQPEDFATYYCQ QFNGYPLTFGGGTKVDIK (SEQ IDNO: 15) HC-9 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-9 hKappaAIRMTQSPSSLSASVGDRVTITCQASQGIRNDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ FNSYPLTFGGGTKLEIK (SEQ IDNO: 16) HC-10 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-10 hKappaNIQMTQSPSSLSTSVGDRVTITCRASQGIGTSLAWYQQKPGKPPKLLIYDASSLESGVPSRLSGSGSGTDFTLTISSLQPEDFATYYCQ QSNSYPITFGQGTRLEIK (SEQ IDNO: 17) HC-11 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-11 hKappaAIQLTQSPSSLSASVGDRVTITCRASQSIGDYLTWYQQKPGKAPKVLIYGASSLQSGVPPRFSGSGSGTDFTLTVSSLQPEDFATYYCQ QLNSYPLTFGGGTKLEIK (SEQ IDNO: 18) HC-12 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-12 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVRSTLAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNGYPLTFGQGTRLEIK (SEQ IDNO: 19) HC-13 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-13 hKappaDIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKLEIK (SEQ IDNO: 20) HC-14 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-14 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGISSFLAWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSASGTDFTLTISSLQPEDFATYYCQQ LNGYPLTFGGGTKVEIK (SEQ IDNO: 21) HC-15 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-15 hKappaAIQLTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGIGPKLLIYDASTLESGVPARFSGSGSRTDFTLTITSLQPEDFATYYCQQ FNGYPLTFGGGTKLEIK (SEQ IDNO: 22) HC-16 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-16 hKappaAIQLTQSPSSLSASVGDRVTITCRASQGITSALAWYQEKPGKAPNLLIYDASSLESGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQ LNSYPLTFGGGTKVDIK (SEQ IDNO: 23) HC-17 hIgG1 QIQLVQSGPELRKPGESVKISCKASGYTFTDYAMYWVKQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSLEASANTANLQISNLKNEDTATYFCARARGLVDDYVMDAWGQGTSVTVSS (SEQ ID NO: 24) LC-17 hLambdaSYELIQPPSASVTLGNTVSLTCVGDELSKRYAQWYQQKPDKTIVSVIYKDSERPSGISDRFSGSSSGTTATLTIHGTLAEDEADYYCLST YSDDNLPVFGGGTKLTVL (SEQID NO: 25) HC-18 hIgG1 EVQLQQYGAELGKPGTSVRLSCKVSGYNIRNTYIHWVNQRPGEGLEWIGRIDPTNGNTISAEKFKTKATLTADTSSHTAYLQFSQLKSDDTAIYFCALNYEGYADYWGQGVMVTGSS (SEQ ID NO: 26) LC-18 hKappaDIQMTQSPSFLSASVGDRVTINCKASQNINKYLNWYQQKVGEAPKRLIFKTNSLQTGIPSRFSGSGSGTDYTLTISSLQTEDVATYFCFQ YNIGYTFGAGTKVELK (SEQ IDNO: 27) HC-19 hIgG1 EVQLQESGPGLVKPSQSLSLTCSVTGYSISSNYRWNWIRKFPGNKVEWMGYINSAGSTNYNPSLKSRISMTRDTSKNQFFLQVNSVTTEDTATYYCARSLRGYITDYSGFFDYWGQGVMVTVSS (SEQ ID NO: 28) LC-19 hKappaDIRMTQSPASLSASLGETVNIECLASEDIFSDLAWYQQKPGKSPQLLIYNANSLQNGVPSRFSGSGSGTRYSLKINSLQSEDVATYFCQ QYKNYPLTFGSGTKLEIK (SEQ IDNO: 29) HC-20 hIgG1 EVQLQQYGAELGKPGTSVRLSCKLSGYKIRNTYIHWVNQRPGKGLEWIGRIDPANGNTIYAEKFKSKVTLTADTSSNTAYMQLSQLKSDDTALYFCAMNYEGYEDYWGQGVMVTVSS (SEQ ID NO: 30) LC-20 hKappaDIQMTQSPSFLSASVGDSVTINCKASQNINKYLNWYQQKLGEAPKRLIHKTDSLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCFQ YKSGFMFGAGTKLELK (SEQ IDNO: 31) HC-21 hIgG1 QIQLVQSGPELKKPGESVKISCKASGYTFTDYAVYWVIQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSLETSASTANLQISNLKNEDTATYFCARGAGMTKDYVMDAWGRGVLVTVS (SEQ ID NO: 32) LC-21 hLambdaSYELIQPPSASVTLGNTVSLTCVGDELSKRYAQWYQQKPDKTIVSVIYKDSERPSDISDRFSGSSSGTTATLTIHGTLAEDEADYYCLST YSDDNLPVFGGGTKLTVL (SEQID NO: 33) HC-22 hIgG1 QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYLVHWVRQPPGKTLEWVGLMWNDGDTSYNSALKSRLSISRDTSKSQVFLKMHSLQAEDTATYYCARESNLGFTYWGHGTLVTVSS (SEQ ID NO: 34) LC-22 hKappaDIQMTQSPASLSASLEEIVTITCKASQGIDDDLSWYQQKPGKSPQLLIYDVTRLADGVPSRFSGSRSGTQYSLKISRPQVADSGIYYCLQ SYSTPYTFGAGTKLELK (SEQ IDNO: 35) HC-23 hIgG1 EVQLQQYGAELGKPGTSVRLSCKVSGYNIRNTYIHWVHQRPGEGLEWIGRIDPTNGNTISAEKFKSKATLTADTSSNTAYMQFSQLKSDDTAIYFCAMNYEGYADYWGQGVMVTVSS (SEQ ID NO: 36) LC-23 hKappaDIQMTQSPSFLSASVGDRLTINCKASQNINKYLNWYQQKLGEAPKRLIFKTNSLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCFQ YNIGFTFGAGTKLELK (SEQ IDNO: 37) HC-24 hIgG1 EVQLVESGGGLVQSGRSLKLSCAASGFTVSDYYMAWVRQAPTKGLEWVATINYDGSTTYHRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCARHGDYGYHYGAYYFDYWGQGVMVTVSS (SEQ ID NO: 38) LC-24 hKappaDIVLTQSPALAVSLGQRATISCRASQTVSLSGYNLIHWYQQRTGQQPKLLIYRASNLAPGIPARFSGSGSGTDFTLTISPVQSDDIATYYC QQSRESWTFGGGTNLEMK (SEQID NO: 39) HC-25 hIgG1 QIQLVQSGPELKKPGESVKISCKASGYTFTDYAIHWVKQAPGQGLRWMAWINTETGKPTYADDFKGRFVFSLEASASTAHLQISNLKNEDTATFFCAGGSHWFAYWGQGTLVTVSS (SEQ ID NO: 40) LC-25 hLambdaSYELIQPPSASVTLENTVSITCSGDELSNKYAHWYQQKPDKTILEVIYNDSERPSGISDRFSGSSSGTTAILTIRDAQAEDEADYYCLSTF SDDDLPIFGGGTKLTVL (SEQ IDNO: 41) HC-26 hIgG1 QIQLVQSGPELKKPGESVKISCKASGYTFTDYAVYWVIQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSLETSASTANLQISNLKNEDTATYFCARGAGMTKDYVMDAWGRGVLVTVS (SEQ ID NO: 32) LC-26 hLambdaSYELIQPPSTSVTLGNTVSLTCVGNELPKRYAYWFQQKPDQSIVRLIYDDDRRPSGISDRFSGSSSGTTATLTIRDAQAEDEAYYYCHS TYTDDKVPIFGGGTKLTVL (SEQID NO: 42) HC-27 hIgG1 EVQLVESGGGLVQPGRSMKLSCKASGFTFSNYDMAWVRQAPTRGLEWVASISYDGITAYYRDSVKGRFTISRENAKSTLYLQLVSLRSEDTATYYCTTEGGYVYSGPHYFDYWGQGVMVTVSS (SEQ ID NO: 43) LC-27 hKappaDIQMTQSPSSMSVSLGDTVTITCRASQDVGIFVNWFQQKPGRSPRRMIYRATNLADGVPSRFSGSRSGSDYSLTISSLESEDVADYHC LQYDEFPRTFGGGTKLELK (SEQ IDNO: 44) HC-28 hIgG1 EVQLQQYGAELGKPGTSVRLSCKVSGYKIRNTYIHWVNQRPGKGLEWIGRIDPANGNTIYAEKFKSKVTLTADTSSNTAYMQLSQLKSDDTALYFCAMNYEGYEDYWGQGVMVTVSS (SEQ ID NO: 45) LC-28 hKappaDIQMTQSPSFLSASVGDSVTINCKASQNINKYLNWYQQKLGEAPKRLIHKTNSLQPGFPSRFSGSGSGTDYTLTISSLQPEDVAAYFCF QYNSGFTFGAGTKLELK (SEQ IDNO: 46) HC-29 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWMNPHSGDTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 47) LC-29 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGNELGWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QYDNLPLTFGQGTKVEIK (SEQ IDNO: 48) HC-30 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGWINPNSGDTNYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 49) LC-30 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPLTFGGGTKVEIK (SEQ IDNO: 50) HC-31 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYEGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 51) LC-31 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASELETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPITFGQGTKVEIK (SEQ IDNO: 52) HC-32 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWLNPSGGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYDGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 53) LC-32 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPLTFGGGTKVEIK (SEQ IDNO: 54) HC-33 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMKLSSLRSEDTAVYYCARHGRGYEGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 55) LC-33 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANGFPLTFGGGTKVEIK (SEQ IDNO: 56) HC-34 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGIINPSGGNTNYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYNAYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 57) LC-34 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QVNGYPLTFGGGTKVEIK (SEQ IDNO: 58) HC-35 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGVINPTVGGANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYNEYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 59) LC-35 hKappaDIQMTQSPSSLSASVGDRVTITCQASQDISDYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QGNSFPLTFGGGTKLEIK (SEQ IDNO: 60) HC-36 hIgG1 QVQLVQSGAEVKKLGASVKVSCKASGYTFSSYYMHWVRQAPGQGLEWMGVINPNGAGTNFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYEGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 61) LC-36 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPLTFGGGTKVEIK (SEQ IDNO: 50) HC-37 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMHWVRQAPGQGLEWMGWINPTGGGTNYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYEGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 62) LC-37 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDVSWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLSGYPITFGQGTKLEIK (SEQ IDNO: 63) HC-38 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGMINPSGGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYNDYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 64) LC-38 hKappaDIQMTQSPSSLSASVGDRVTITCRASQSISDWLAWYQQKPGKAPKLLIYEASNLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPYTFGQGTKVEIK (SEQ IDNO: 65) HC-39 hIgG1 QVQLVQSGAEVKKPGASVKVSCKASGYIFSAYYIHWVRQAPGQGLEWMGIINPSGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYGGYEGAFDIWDQGTLVTVSSAS (SEQ ID NO: 66) LC-39 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGDYVAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPITFGQGTRLEIK (SEQ IDNO: 67) HC-40 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTSYWIGWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISVDKSNSTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 68) LC-40 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTYFTLTISSLQPEDFATYYCQ QGASFPITFGQGTKVEIK (SEQ IDNO: 69) HC-41 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGSSFPNSWIAWVRQMPGKGLEWMGIIYPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLEASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 70) LC-41 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNSYPLTFGGGTKVEIK (SEQ IDNO: 71) HC-42 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIGWVRQMPGKGLEWMGIMYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNAYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 72) LC-42 hKappaDIQMTQSPSSLSASVGDRVTITCRASQSINNWLAWYQQKPGKAPKLLIYDAFILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQ LNSYPLTFGPGTKVDIK (SEQ IDNO: 73) HC-43 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNWIAWVRQMPGKGLEWMGIIYPGDSETRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYYGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 74) LC-43 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGISDNLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QAISFPLTFGQGTKVEIK (SEQ IDNO: 75) HC-44 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYNFTSYWIGWVRQMPGKGLEWMGVIYPDDSETRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 76) LC-44 hKappaDIQMTQSPSSLSASVGDRVTITCRASRDIRDDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPLTFGGGTKVEIK (SEQ IDNO: 77) HC-45 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYTFNTYIGWVRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQVTISADKAISTAYLQWSSLKASDTAMYYCARHSRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 78) LC-45 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPVTFGQGTKVEIK (SEQ IDNO: 79) HC-46 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYNFTTYWIGWVRQMPGKGLEWMGIIHPADSDTRYNPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 80) LC-46 hKappaDIQMTQSPSSLSASVGDRVTITCRVSQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPLTFGGGTKVEIK (SEQ IDNO: 81) HC-47 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFSNYWIAWVRQMPGKGLEWMGIIYPDNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYDGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 82) LC-47 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRSDLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPLSFGQGTKVEIK (SEQ IDNO: 83) HC-48 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFASYWIGWVRQMPGKGLEWMGITYPGDSETRYNPSQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYGGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 84) LC-48 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPLTFGGGTKVEIK (SEQ IDNO: 85) HC-49 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSSAS (SEQ ID NO: 86) LC-49 hKappaDIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QTNSFPLTFGQGTRLEIK (SEQ IDNO: 87) HC-74 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-74 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 88) HC-75 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-75 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 89) HC-76 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-76 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 90) HC-77 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-77 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIK (SEQ IDNO: 91) HC-78 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-78 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIK (SEQ IDNO: 92) HC-79 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-79 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIK (SEQ IDNO: 93) HC-80 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-80 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIK (SEQ IDNO: 94) HC-81 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-81 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 95) HC-82 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-82 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 96) HC-83 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-83 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 97) HC-84 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-84 hKappaDIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK (SEQ IDNO: 97) HC-245 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQGTLVTVSS (SEQ ID NO: 98) LC-245 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNGYPLTFGQGTRLEIK (SEQ IDNO: 99) HC-246 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-246 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QFNGYPLTFGQGTRLEIK (SEQ IDNO: 99) HC-247 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS (SEQ ID NO: 7) LC-247 hKappaDIQMTQSPSSLSASVGDRVTITCRASRGISDYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPITFGQGTRLEIK (SEQ IDNO: 100) HC-248 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQGTLVTVSS (SEQ ID NO: 98) LC-248 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPLTFGQGTRLEIK (SEQ IDNO: 101) HC-249 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQGTLVTVSS (SEQ ID NO: 98) LC-249 hKappaDIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QLNGYPLTFGQGTRLEIK (SEQ IDNO: 102)

The nucleic acid sequences corresponding to the heavy and light chainregions of certain sequences described above are provided in Table 2.

TABLE 2 Heavy and light chain anitbody variable region nucleic acidsequences HC-1 hIgG1 CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-1 hKappaGCCATTCAACTTACACAAAGTCCGAGTAGTCTCAGCGCGAGCGTCGGGGACCGGGTAACCATAACTTGCCGAGCCAGCCAGGGCGTCTCTAGCGCATTGGCATGGTATCAACAAAAACCTGGAAAGGCTCCCAAGCTCCTCATTTACGATGCTAGCTCCCTTGAATCTGGCGTACCATCCCGCTTTAGTGGCAGTGGGTCTGGAACAGACTTTACTCTTACAATATCATCCCTGCAACCAGAAGATTTTGCTACCTACTACTGTCAACAGTTTAATAGTTACCCACTCACATTCGGCGGGGGTACGAAAGTAGAAATAAAGCGAACCGTGGCTGCGCCTAGCGTCTTTATCTTTCCCCCGAGCGATGAACAGTTGAAATCAGGAACTGCTTCTGTGGTATGTTTGCTTAATAATTTTTACCCACGGGAAGCAAAAGTGCAGTGGAAAGTAGACAATGCGCTCCAGTCCGGCAATTCTCAAGAGAGTGTGACTGAACAGGATTCTAAGGATAGCACTTATTCACTGTCAAGTACCTTGACATTGTCAAAGGCGGACTATGAGAAACATAAGGTTTACGCCTGTGAGGTAACACACCAAGGGCTCAGCTCACCTGTTACGAAATCCTTCAATAGGGGC GAGTGT (SEQ ID NO:104) HC-2 hIgG1 CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-2 hKappaGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAACTGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 105) HC-3 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-3 hKappaGCCATCCGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGCCTGGTATCAGCAGAAACCAGGGAAAACTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGCTAA(SEQ ID NO: 106) HC-4 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-4 hKappaGCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTGACTTTCGGCGGAGGGACCAAAGTGGATATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 107) HC-5 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-5 hKappaAACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGCCATTAGCGATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAGTTACCCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 108) HC-6 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-6 hKappaGCCATCCGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGGGACAGAGTCATTATCGCTTGCCGGGCAAGTCAGGGCATCGGCGGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAATGCTCCTAAGGTCCTGGTCTATGATGCCTCCACTTTGGAAAGTGGGGTCCCATCACGGTTCAGCGGCGGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 109) HC-7 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-7 hKappaGACATCGCGATGACCCAGTCTCCACCCTCCCTGTCTGCATTTGTAGGGGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTATCAGTTCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATG CCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCCGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 110) HC-8 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-8 hKappaGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAAGCAGGGAAAGCTCCTAAAGTCCTGATCTCTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAGCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATGGTTACCCGCTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 111) HC-9 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-9 hKappaGCCATCCGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTAGAAATGATTTAGGCTG GTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 112) HC-10 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-10 hKappaAACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTACATCCGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTGGCACTTCTTTAGCCTG GTATCAGCAGAAGCCAGGGAAGCCTCCTAAGTTACTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGCTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTAATAGTTATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 113) HC-11 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-11 hKappaGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGGCGACTATTTGACTTGGTATCAGCAGAAACCAGGCAAAGCCCCTAAGGTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCACCAAGGTTCAGTGGCAGTGGTTCTGGGACAGATTTCACTCTCACCGTCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAGTTACCCCCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGCTAA (SEQID NO: 114) HC-12 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-12 hKappaGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACGTGCCGGGCAAGTCAGGGCGTTAGGAGTACTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCATTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATGGTTACCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGCTAA(SEQ ID NO: 115) HC-13 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-13 hKappaGATATTGTGATGACTCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGCTAA (SEQID NO: 116) HC-14 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-14 hKappaGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTTTTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGCATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATGGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGCTAA(SEQ ID NO: 117) HC-15 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-15 hKappaGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTGGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGATAGGTCCTAAGCTCCTGATCTATGATGCCTCAACTTTGGAAAGTGGGGTCCCAGCAAGGTTCAGCGGCAGTGGATCTAGGACAGATTTCACTCTCACCATCACCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATGGTTACCCTCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGCTAA (SEQID NO: 118) HC-16 hIgG1CAGGTGCAGCTGGTGCAGAGCGGTGCGGCGGTGAAAAAACCTGGCGAAAGCCTGAAAATTAGCTGCAAAGGCAGCGGCTATCGTTTTACCACCTATTGGATTGGCTGGGTGCGTCAGATGCCGGGCAAAGGACTGGAATGGATGGGCATTATCTATCCGGGCGATAGCGATACCCGTTACAGCCCTAGCTTTCAGGGGCAGGTGACCATTAGCGCGGGAAAAAGCATTAGCACCGCGTATCTGCAGTGGAGCAGCTTAAAAGCGAGCGACACCGCGATGTATTATTGCGCGCGTCATGGCCGTGGCTATAATGGCTATGAAGGCGCGTTTGATATTTGGGGCCAGGGGACTATGGTTACCGTGAGCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCG GATAGTAA (SEQ IDNO: 103) LC-16 hKappaGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTACCAGTGCTTTAGCCTGGTATCAGGAGAAACCAGGGAAAGCTCCTAACCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATATGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAACGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGCTAA (SEQID NO: 119) HC-17 hIgG1cagatccagttggtacagtctggacctgagctgaggaagcctggcgagtcagtgaagatctcctgcaaggcttctggatataccttcacagactatgcaatgtattgggtgaaacaggctccaggaaagggcttgaagtggatgggctggatcaacacctatactgggaagccaacatatgctgatgacttcaaaggacgatttgtcttctctttggaagcctctgccaacactgcaaatttgcagatcagcaacctcaaaaatgaggacacggctacatatttctgtgcaagagcccgcggattagtcgatgactatgttatggatgcctggggtcaagggacttcagtcactgtctcctct (SEQ ID NO: 120) LC-17 hLambdaagctatgagctgatccaaccaccttcggcatcagtcactctgggaaatactgtctcactcacttgtgtcggagatgaattatcaaaaagatatgctcagtggtatcaacaaaagccagacaagaccattgtgtccgtgatatacaaagatagtgagcggccctcaggcatctctgaccgattctctggttccagctccgggacaacagccactctgacaatccatggcaccctggctgaggatgaggctgattattactgtttgtcaacatatagtgatgataatctccctgttttcggtggtggaaccaagctcactgtccta (SEQ ID NO: 121) HC-18hIgG1gaagtccagctgcagcagtatggggctgagcttgggaaacctgggacctcagtcaggttgtcttgcaaggtttctggctataacattaggaatacctacattcactgggtgaatcagaggcctggagagggcctggaatggataggaaggattgatcctacaaacggaaatactatatctgctgagaaattcaaaaccaaggccacactgactgcagatacatcgtcccacacagcctacttgcagttcagccaactgaaatctgacgacacagcaatctatttttgtgctctgaactatgagggatatgcggattattggggccagggagtcatggtcacaggctcctcc (SEQ ID NO: 122) LC-18 hKappagacatccagatgacccagtctccttcattcctgtctgcatctgtgggagacagagtcactatcaactgcaaagcaagtcagaatattaacaagtacttaaactggtatcagcaaaaggttggagaagctcccaaacgcctgatatttaagacaaacagtttgcaaacgggcatcccatcaaggttcagtggcagtggatctggaacagattatacactcaccatcagcagcctgcagactgaagatgttgccacatatttctgctttcagtataacattgggtacacgtttggagctgggaccaaggtggagctgaaa (SEQ ID NO: 123) HC-19 hIgG1gaggtgcagcttcaggagtcaggacctggccttgtgaaaccctcacagtcactctccctcacctgttcggtcactggatactccatttccagtaattatagatggaactggatccggaagttcccaggaaataaagtggagtggatgggatatataaacagtgcaggcagtactaactacaatccgtctctcaaaagtcgaatctccatgactagagacacatccaagaatcagttcttcctgcaggtgaactctgtaacaactgaggacacagccacttattactgtgcgagatccctaagagggtatattacggattattcaggcttctttgattactggggccaaggagtcatggtcacagtctcctca (SEQ ID NO: 124) LC-19 hKappagatatccggatgacacagtctccagcttccctgtctgcatctctgggagagactgtcaacatcgaatgtctagcaagtgaggacattttcagtgatttagcatggtatcagcagaagccagggaaatctcctcaactcctgatctataatgcaaatagcttgcaaaatggggtcccttcacggtttagtggcagtggatctggcacacggtattctctcaaaataaacagcctgcaatctgaagatgtcgcgacttatttctgtcaacaatataagaattatccgctcacgttcggttctgggaccaagctggagatcaaa (SEQ ID NO: 125) HC-20 hIgG1gaagtccagctgcagcagtatggggctgagcttgggaaacctgggacctcagtcaggttgtcttgcaagctttctggctataagattaggaatacctacatacactgggtgaatcagaggcctggaaagggcctggaatggattgggaggattgatcctgcaaatggaaatactatctatgctgagaagttcaaaagcaaggttacactgactgcagatacatcgtccaacacagcctacatgcaactcagccaactgaaatctgacgacacagcactctatttttgtgctatgaactacgaagggtatgaggattactggggccaaggagtcatggtcacagtctcctca (SEQ ID NO: 126) LC-20 hKappagacatccagatgacccagtctccttcattcctgtctgcatctgtgggagacagcgtcactatcaactgcaaagcaagtcagaatattaacaagtacttaaattggtatcagcaaaagcttggagaagctcccaaacgcctgatacataaaacagacagtctgcaaacgggcatcccatcaaggttcagtggcagtggatctggtacagattacacactcaccatcagcagcctgcagcctgaagatgttgccacatacttctgctttcagtataagagtgggttcatgtttggagctgggaccaagctggaactgaaa (SEQ ID NO: 127) HC-21 hIgG1cagatccagttggtacagtctggacctgagctgaagaagcctggagagtcagtgaagatctcctgcaaggcctctgggtataccttcacagactatgcagtgtactgggtgatacaggctccaggaaagggcttgaagtggatgggctggatcaacacctatactgggaagccaacatatgccgatgacttcaaaggacggtttgtcttctctttggaaacctctgccagcactgcaaatttgcagatcagcaacctcaaaaatgaggacacggctacatatttctgtgcaagaggagcgggcatgactaaggactatgttatggatgcctggggtcgaggggttttagtcactgtctcctca (SEQ ID NO: 128) LC-21 hLambdaagctatgagctgatccaaccaccttcggcgtcagtcactctgggaaatactgtctcactcacttgtgtcggagatgaattatcaaaaagatatgctcagtggtatcaacaaaagccagacaagaccattgtgtccgtgatatacaaagatagtgagcggccctcagacatctctgaccgattctctggttccagctccgggacaacagccactctgacaatccatggcaccctggctgaggatgaggctgattattactgtttgtcaacatatagtgatgataatctccctgttttcggtggtggaaccaagctcactgtccta (SEQ ID NO: 129) HC-22hIgG1caggtgcagctgaaggagtcaggacctggcctggtgcagccctcacagaccctgtctctcacctgcactgtctctggattctcattaaccagctatcttgttcactgggttcgacagcctccaggaaaaactctggagtgggtgggattaatgtggaatgatggagacacatcatataattcagctctcaaatcccgactgagcatcagcagggacacctccaagagccaagttttcttaaagatgcacagtcttcaagctgaggacacagccacttactactgtgccagagagagcaacttgggatttacttactggggccacggcactctggtcactgtctcttca (SEQ ID NO: 130) LC-22 hKappagacatccagatgacacagtctcctgcctccctgtctgcttctctggaagaaattgtcaccatcacctgcaaggcaagccagggcattgatgatgacttatcatggtatcagcagaaaccagggaaatctcctcagctcctgatctatgatgtaaccagattggcagatggggtcccatcacggttcagcggcagtagatctggcacacagtattctcttaagatcagcagaccacaggttgctgattctggaatctattactgtctgcagagttacagtactccgtacacgtttggagctgggaccaagctggaactgaaa (SEQ ID NO: 131) HC-23hIgG1gaagtccagctgcagcagtatggggctgagcttgggaaacctgggacctcagtcaggttgtcttgcaaggtttctggctataacattaggaatacctacattcactgggtgcatcagaggcctggagagggcctggaatggataggaaggattgatcctacaaacggaaatactatatctgctgagaagttcaaaagcaaggccacactgactgcagatacatcgtccaatacagcctacatgcagttcagccaactgaaatctgacgacacagcaatctatttttgtgctatgaactacgaagggtatgcggattattggggccaaggagtcatggtcacagtctcctcc (SEQ ID NO: 132) LC-23 hKappagacatccagatgacccagtctccttcattcctgtctgcatctgtgggagacagactcactatcaactgcaaagcaagtcagaatattaacaagtacttaaactggtatcagcaaaagcttggagaagctcccaaacgcctgatatttaagacaaacagtttgcaaacgggcatcccatcaaggttcagtggcagtggatctggaacagattacacactcaccatcagcagcctgcagcctgaagatgttgccacatatttctgctttcagtataacattgggttcacgtttggagctgggaccaagctggagctgaaa (SEQ ID NO: 133) HC-24 hIgG1gaggtgcagctggtggagtctggtggaggcttagtgcagtctggaaggtccctaaaactctcctgtgcagcctcaggattcactgtcagtgactattacatggcctgggtccgccaggctccaacgaaggggctggagtgggtcgcaaccattaattatgatggtagtaccacttaccatcgagactccgtgaagggccgattcactatctccagggataatgcaaaaagcaccctatacctgcaaatggacagtctgcggtctgaggacacggccacttattactgtgcaagacatggggactatgggtatcactacggggcctattattttgattactggggccaaggagtcatggtcacagtctcctca (SEQ ID NO: 134) LC-24 hKappagacattgtcttgacccagtctcctgctttggctgtgtctctggggcagagggccactatctcctgtagggccagccagactgtcagtttatctggatataatcttatacactggtaccaacagagaacaggacagcaacccaaactcctcatctatcgtgcatccaatctagcacctgggatccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaccatcagccctgtgcagtctgatgatattgcaacctattactgtcagcagagtagggagtcgtggacgttcggtggaggcaccaacttggaaatgaag (SEQ ID NO: 135) HC-25hIgG1cagatccagttggtacagtctggacctgagctgaagaagcctggagagtcagtgaagatctcctgcaaggcttctgggtataccttcacagactatgcaatacactgggtgaaacaggctccaggacagggcttgaggtggatggcctggatcaacaccgaaactgggaagcctacatatgctgatgacttcaaaggacggtttgtcttctctttggaggcctctgccagcactgcacatttgcagatcagcaacctcaaaaatgaggacacggctacatttttctgtgcaggcgggtcccattggtttgcttactggggccaaggcactctggtcactgtctcttca (SEQ ID NO: 136) LC-25 hLambdaagctatgagctgatccaaccaccttcagcatctgtcactctggaaaatactgtctcaatcacttgttctggagatgaattatcaaacaaatatgctcattggtatcaacaaaagccagacaagaccattttggaagtgatctacaacgatagtgagcggccctcaggcatctctgaccgattctctgggtccagctcagggacaacagccattctcacaatccgtgatgcccaggctgaggatgaggctgattattactgtttgtcaacatttagtgatgatgatctccctattttcggtggtggcaccaagctcactgtccta (SEQ ID NO: 137) HC-26hIgG1cagatccagttggtacagtctggacctgagctgaagaagcctggagagtcagtgaagatctcctgcaaggcctctgggtataccttcacagactatgcagtgtactgggtgatacaggctccaggaaagggcttgaagtggatgggctggatcaacacctatactgggaagccaacatatgccgatgacttcaaaggacggtttgtcttctctttggaaacctctgccagcactgcaaatttgcagatcagcaacctcaaaaatgaggacacggctacatatttctgtgcaagaggagcgggcatgactaaggactatgttatggatgcctggggtcgaggggttttagtcactgtctcctca (SEQ ID NO: 128) LC-26 hLambdaagctatgagctgatccaaccaccttcaacatcagtcactctgggaaatactgtctcactcacctgtgttggaaatgaattaccaaaaagatatgcttattggtttcaacaaaagccagaccagtccattgtgagactgatatatgacgatgacaggcggccctcaggcatctctgaccgattctctgggtccagctctgggacaacagccactctgacaatccgtgacgcccaggctgaggatgaggcttattattactgtcactcaacatatactgatgataaagtccctattttcggtggtggaaccaagctcactgtccta (SEQ ID NO: 138) HC-27hIgG1gaggtgcagctggtggagtctgggggaggcttagtgcagcctggaaggtccatgaaactctcctgtaaggcctcaggattcactttcagtaactatgacatggcctgggtccgccaggctccaacgaggggtctggagtgggtcgcatccattagttatgatggtattaccgcttactatcgagactccgtgaagggccgattcactatctccagagagaatgcaaaaagcaccctatacctgcaattggtcagtctgagatctgaggacacggccacttattactgtacaacagaggggggttatgtgtactccggaccacactactttgattactggggccaaggagtcatggtcacagtctcctca (SEQ ID NO: 139) LC-27 hKappagacattcagatgacccagtctccatcctccatgtctgtgtctctgggagacacagtcactattacttgccgggcaagtcaggacgttgggatttttgtaaattggttccagcagaaaccagggagatctcctaggcgtatgatttatcgtgcaacgaacttggcagatggggtcccatcaaggttcagcggcagtaggtctggatcagattattctctcaccatcagcagcctggagtctgaagatgtggcagactatcactgtctacagtatgatgagtttcctcggacgttcggtggaggcaccaagctggaattgaaa (SEQ ID NO: 140) HC-28 hIgG1gaagtccagctgcagcagtatggggctgagcttgggaaacctgggacctcagtcaggttgtcttgcaaggtttctggctataagattaggaatacctacatacactgggtgaatcagaggcctggaaagggcctggaatggatagggaggattgatcctgcaaatggaaatactatatatgctgagaagttcaaaagcaaggttacactgactgcagatacatcgtccaacacagcctacatgcaactcagccaactgaaatctgacgacacagcactctatttttgtgctatgaactacgaagggtatgaggattactggggccaaggagtcatggtcacagtctcctca (SEQ ID NO: 141) LC-28 hKappagacatccagatgacccagtctccttcattcctgtctgcatctgtgggagacagcgtcactatcaactgcaaagcaagtcagaatattaataagtatttaaactggtatcagcaaaagcttggagaagctcccaaacgcctgatacataaaacaaacagtttgcaaccgggcttcccatcaaggttcagtggcagtggatctggtacagattacacactcaccatcagcagcctgcagcctgaagatgttgccgcatatttctgctttcagtataacagtgggttcacgtttggagctgggaccaagctggaactgaaa (SEQ ID NO: 142)

As described below, an scFV phage display library screen of humanantibodies was performed to identify novel anti-CD117 antibodies, andfragments thereof, having therapeutic use. Antibodies 85 (Ab85), 86(Ab86), 87 (Ab87), 88 (Ab88), and 89 (Ab89), among others, wereidentified in this screen.

The heavy chain variable region (VH) amino acid sequence of Ab85 isprovided below as SEQ ID NO: 143. The VH CDR amino acid sequences ofAb85 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI(VH CDR3; SEQ ID NO: 147).

Ab85 VH sequence (SEQ ID NO: 143)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab85 isprovided below as SEQ ID NO 144. The VL CDR amino acid sequences of Ab85are underlined below and are as follows: RSSQGIRSDLG (VL CDR1; SEQ IDNO: 148); DASNLET (VL CDR2; SEQ ID NO: 149); and QQANGFPLT (VL CDR3; SEQID NO: 150).

Ab85 VL sequence (SEQ ID NO: 144)DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTF GGGTKVEIKAntibody HC-86/LC-86 (Ab86)

The heavy chain variable region (VH) amino acid sequence of Ab86 isprovided below as SEQ ID NO: 151. The VH CDR amino acid sequences Ab86are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IIYPGDSDIRYSPSLQG (VH CDR2; SEQ ID NO: 153); and HGRGYNGYEGAFDI(VH CDR3; SEQ ID NO: 3).

Ab86 VH sequence (SEQ ID NO: 151)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDIRYSPSLQGQVTISVDTSTSTAYLQWNSLKPSDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab86 isprovided below as SEQ ID NO 152. The VL CDR amino acid sequences of Ab86are underlined below and are as follows: RASQGIGDSLA (VL CDR1; SEQ IDNO: 154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3; SEQID NO: 155).

Ab86 VL sequence (SEQ ID NO: 152)DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITF GQGTKVEIKAntibody HC-87/LC-87 (Ab87)

The heavy chain variable region (VH) amino acid sequence of Ab87 isprovided below as SEQ ID NO: 143. The VH CDR amino acid sequences ofAb87 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI(VH CDR3; SEQ ID NO: 147).

Ab87 VH sequence (SEQ ID NO: 143)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab87 isprovided below as SEQ ID NO 156. The VL CDR amino acid sequences of Ab87are underlined below and are as follows: RASQGIRNDLG (VL CDR1; SEQ IDNO: 157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3; SEQID NO: 155).

Ab87 VL sequence (SEQ ID NO: 156)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-88/LC-88 (Ab88)

The heavy chain variable region (VH) amino acid sequence of Ab88 isprovided below as SEQ ID NO: 158. The VH CDR amino acid sequences ofAb88 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IIYPGDSLTRYSPSFQG (VH CDR2; SEQ ID NO: 159); and HGRGYNGYEGAFDI(VH CDR3; SEQ ID NO: 3).

Ab88 VH sequence (SEQ ID NO: 158)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSLTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab88 isprovided below as SEQ ID NO: 156. The VL CDR amino acid sequences ofAb88 are underlined below and are as follows: RASQGIRNDLG (VL CDR1; SEQID NO: 157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3;SEQ ID NO: 155).

Ab88 VL sequence (SEQ ID NO: 156)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-89/LC-89 (Ab89)

The heavy chain variable region (VH) amino acid sequence of Ab89 isprovided below as SEQ ID NO: 160. The VH CDR amino acid sequences ofAb89 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGRGYNGYEGAFDI (VHCDR3; SEQ ID NO: 3).

Ab89 VH sequence (SEQ ID NO: 160)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab89 isprovided below as SEQ ID NO: 152. The VL CDR amino acid sequences ofAb89 are underlined below and are as follows: RASQGIGDSLA (VL CDR1; SEQID NO: 154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3;SEQ ID NO: 155).

Ab89 VL sequence (SEQ ID NO: 152)DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-249/LC-249 (Ab249)

The heavy chain variable region (VH) amino acid sequence of Ab249 isprovided below as SEQ ID NO: 98. The VH CDR amino acid sequences ofAb249 are underlined below and are as follows: TSWIG (VH CDR1; SEQ IDNO: 186); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGLGYNGYEGAFDI(VH CDR3; SEQ ID NO: 187).

Ab249 VH sequence (SEQ ID NO: 98)EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab249 isprovided below as SEQ ID NO: 102. The VL CDR amino acid sequences ofAb249 are underlined below and are as follows: RASQGIGSALA (VL CDR1; SEQID NO: 188); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPLT (VL CDR3;SEQ ID NO: 189).

Ab249 VL sequence (SEQ ID NO: 102)DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQ GTRLEIK

Human antibodies Ab85 and Ab249 were both derived from antibody CK6,which is an antagonist anti-CD117 antibody. A comparison of the aminoacid sequences of the variable regions of Ab85 and Ab249 versus CK6 isshown in FIGS. 11A to 11D (where the CDRs are also designated). Bothantibodies have improved properties, e.g., improved bindingcharacteristics, over CK6.

CK6 includes a potential deamidation site in the CDR3 domain of theheavy chain variable region. While advantageous to remove for futureproduction of the antibody, the position of the asparagine presents asignificant challenge. The potential deamidation site was successfullyremoved, however, in the Ab85 heavy chain CDR3 such that the antibody(having Ab85 heavy and light chain CDRs) was able to maintain a highaffinity level specificity for human CD117 and the ability tointernalize. Further, Ab85 has an improved off rate relative to itsparent.

Thus, in certain embodiments, an anti-CD117 antibody comprises a heavychain comprising a CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ IDNos: 145, 146, and 147, and a light chain comprising a CDR set as setforth in SEQ ID Nos: 148, 149, and 150, internalizes in cells expressingCD117, and has a k_(off) rate of 5×10⁻⁴ s⁻¹ or less as measured by BLI.

As described in Jain et al. (2017) PNAS 114 (5) 944-949, while theactivity of an antibody is key for developing it as a therapeutic, whatis often overlooked is the “developability” of an antibody formanufacturing. Identifying an antibody that can achieve both therapeuticand superior biophysical characteristics is challenging. Indeed,biophysical properties of an antibody are essential for antibodies beingused for therapeutic purposes. Biophysical testing of Ab 85 indicatedthat it is a particularly stable antibody. For example, a population ofAb85 antibodies maintains a low level of acid variants even at highertemperatures over time (see FIGS. 13A and 13B). This was true even inrelation to other CK6-derived antibodies. Thus, in certain embodiments,included in the invention is a composition comprising less than 20%acidic variants as determined by capillary electrophoresis followingstorage at 25 degrees Celsius for seven days, wherein the antibody is anIgG antibody that specifically binds to CD117 and comprises a heavychain comprising a CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ IDNos: 145, 146, and 147, and a light chain comprising a CDR set as setforth in SEQ ID Nos: 148, 149, and 150.

The anti-CD117 antibodies or binding fragments described herein may alsoinclude modifications and/or mutations that alter the properties of theantibodies and/or fragments, such as those that increase half-life,increase or decrease ADCC, etc., as is known in the art.

In one embodiment, the anti-CD117 antibody, or binding fragment thereof,comprises a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule has an altered affinity for an FcgammaR. Certainamino acid positions within the Fc region are known throughcrystallography studies to make a direct contact with FcγR. Specificallyamino acids 234-239 (hinge region), amino acids 265-269 (B/C loop),amino acids 297-299 (C′/E loop), and amino acids 327-332 (F/G) loop.(see Sondermann et al., 2000 Nature, 406: 267-273). For example, aminoacid substitutions at amino acid positions 234 and 235 of the Fc regionhave been identified as decreasing affinity of an IgG antibody forbinding to an Fc receptor, particularly an Fc gamma receptor (FcγR). Inone embodiment, an anti-CD117 antibody described herein comprises an Fcregion comprising an amino acid substitution at L234 and/or L235, e.g.,L234A and L235A (EU index). Thus, the anti-CD117 antibodies describedherein may comprise variant Fc regions comprising modification of atleast one residue that makes a direct contact with an FcγR based onstructural and crystallographic analysis. In one embodiment, the Fcregion of the anti-CD117 antibody (or Fc containing fragment thereof)comprises an amino acid substitution at amino acid 265 according to theEU index as in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, NH1, MD (1991), expresslyincorporated herein by references. The “EU index as in Kabat” or “EUindex” refers to the numbering of the human IgG1 EU antibody and is usedherein in reference to Fc amino acid positions unless otherwiseindicated.

In one embodiment, the Fc region comprises a D265A mutation. In oneembodiment, the Fc region comprises a D265C mutation.

In some embodiments, the Fc region of the anti-CD117 antibody (orfragment thereof) comprises an amino acid substitution at amino acid 234according to the EU index as in Kabat. In one embodiment, the Fc regioncomprises a L234A mutation. In some embodiments, the Fc region of theanti-CD117 antibody (or fragment thereof) comprises an amino acidsubstitution at amino acid 235 according to the EU index as in Kabat. Inone embodiment, the Fc region comprises a L235A mutation. In yet anotherembodiment, the Fc region comprises a L234A and L235A mutation. In afurther embodiment, the Fc region comprises a D265C, L234A, and L235Amutation.

In certain aspects a variant IgG Fc domain comprises one or more aminoacid substitutions resulting in decreased or ablated binding affinityfor an FcgammaR and/or C1q as compared to the wild type Fc domain notcomprising the one or more amino acid substitutions. Fc bindinginteractions are essential for a variety of effector functions anddownstream signaling events including, but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC). Accordingly, in certain aspects, an antibodycomprising a modified Fc region (e.g., comprising a L234A, L235A, and aD265C mutation) has substantially reduced or abolished effectorfunctions.

Affinity to an Fc region can be determined using a variety of techniquesknown in the art, for example but not limited to, equilibrium methods(e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswamiet al. Analytical Biochemistry, Vol. 373:52-60, 2008; orradioimmunoassay (RIA)), or by a surface plasmon resonance assay orother mechanism of kinetics-based assay (e.g., BIACORE™ analysis orOctet™ analysis (forteBIO)), and other methods such as indirect bindingassays, competitive binding assays fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions. One example of a competitivebinding assay is a radioimmunoassay comprising the incubation of labeledantigen with the antibody of interest in the presence of increasingamounts of unlabeled antigen, and the detection of the antibody bound tothe labeled antigen. The affinity of the antibody of interest for aparticular antigen and the binding off-rates can be determined from thedata by scatchard plot analysis. Competition with a second antibody canalso be determined using radioimmunoassays. In this case, the antigen isincubated with antibody of interest conjugated to a labeled compound inthe presence of increasing amounts of an unlabeled second antibody.

In one embodiment, an anti-CD117 antibody described herein comprises anFc region comprising L234A, L235A, and D265C (EU index). The antibodiesof the invention may be further engineered to further modulate antibodyhalf-life by introducing additional Fc mutations, such as thosedescribed for example in (Dall'Acqua et al. (2006) J Biol Chem 281:23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton etal. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol176: 346-56), (Shields et al. (2001) J Biol Chem 276: 6591-604),(Petkova et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al.(2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) NatBiotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and(Kim et al. (1999) Eur J Immunol 29: 2819-25), and include positions250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplarymutations that may be made singularly or in combination are T250Q,M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K,N434S, N434A, N434H, N434F, H435A and H435R mutations.

Thus, in one embodiment, the Fc region comprises a mutation resulting ina decrease in half life. An antibody having a short half life may beadvantageous in certain instances where the antibody is expected tofunction as a short-lived therapeutic, e.g., the conditioning stepdescribed herein where the antibody is administered followed by HSCs.Ideally, the antibody would be substantially cleared prior to deliveryof the HSCs, which also generally express CD117 but are not the targetof the anti-CD117 antibody, unlike the endogenous stem cells. In oneembodiment, the Fc region comprises a mutation at position 435 (EU indexaccording to Kabat). In one embodiment, the mutation is an H435Amutation.

In one embodiment, the anti-CD117 antibody described herein has a halflife of equal to or less than 24 hours, equal to or less than 22 hours,equal to or less than 20 hours, equal to or less than 18 hours, equal toor less than 16 hours, equal to or less than 14 hours, equal to or lessthan 13 hours, equal to or less than 12 hours, or equal to or less than11 hours. In one embodiment, the half life of the antibody is 11 hoursto 24 hours; 12 hours to 22 hours; 10 hours to 20 hours; 8 hours to 18hours; or 14 hours to 24 hours

Anti-CD117 antibodies that can be used in conjunction with the patientconditioning methods described herein include, for instance, antibodiesproduced and released from ATCC Accession No.

10716 (deposited as BA7.3C.9), such as the SR-1 antibody, which isdescribed, for example, in U.S. Pat. No. 5,489,516, the disclosure ofwhich is incorporated herein by reference as it pertains to anti-CD117antibodies.

In one embodiment, an anti-CD117 antibody described herein comprises anFc region comprising L234A, L235A, D265C, and H435A (EU index).

Additional anti-CD117 antibodies that can be used in conjunction withthe patient conditioning methods described herein include thosedescribed in U.S. Pat. No. 7,915,391, which describes, e.g., humanizedSR-1 antibodies; U.S. Pat. No. 5,808,002, which describes, e.g., theanti-CD117 A3C6E2 antibody, as well as those described in, for example,WO 2015/050959, which describes anti-CD117 antibodies that bind epitopescontaining Pro317, Asn320, Glu329, Val331, Asp332, Lys358, Glu360,Glu376, His378, and/or Thr380 of human CD117; and US 2012/0288506 (alsopublished as U.S. Pat. No. 8,552,157), which describes, e.g., theanti-CD117 antibody CK6, having the CDR sequences of:

-   a CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1);-   a CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID    NO: 2);-   a CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:    3);-   a CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4);-   a CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and-   a CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6)

The heavy chain variable region amino acid sequence of CK6 is providedin SEQ ID NO: 161):

(SEQ ID NO: 161; CDRs are underlined are in bold)QVQLVQSGAAVKKPGESLKISCKGSGYRFT SYWIG WVRQMPGKGLEWMG I IYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCAR HG RGYNGYEGAFDI WGQGTMVTVSS.

The light chain amino acid variable sequence of CK6 is provided in SEQID NO: 162:

(SEQ ID NO: 162; CDRs are underlined and in bold)AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY D ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQFNSYPLT FGG GTKVEIK.

Additional anti-CD117 antibodies and antigen-binding fragments thereofthat may be used in conjunction with the compositions and methodsdescribed herein include those described in US 2015/0320880, such as theclones 9P3, NEG024, NEG027, NEG085, NEG086, and 20376.

The disclosures of each of the foregoing publications are incorporatedherein by reference as they pertain to anti-CD117 antibodies. Antibodiesand antigen-binding fragments that may be used in conjunction with thecompositions and methods described herein include the above-describedantibodies and antigen-binding fragments thereof, as well as humanizedvariants of those non-human antibodies and antigen-binding fragmentsdescribed above and antibodies or antigen-binding fragments that bindthe same epitope as those described above, as assessed, for instance, byway of a competitive CD117 binding assay.

Exemplary antigen-binding fragments of the foregoing antibodies includea dual-variable immunoglobulin domain, a single-chain Fv molecule(scFv), a diabody, a triabody, a nanobody, an antibody-like proteinscaffold, a Fv fragment, a Fab fragment, a F(ab′)₂ molecule, and atandem di-scFv, among others.

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CD117 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CLL-1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CD117 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

In one embodiment, the anti-CD117 antibody, or antigen binding fragmentthereof, comprises variable regions having an amino acid sequence thatis at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosedherein. Alternatively, the anti-CD117 antibody, or antigen bindingfragment thereof, comprises CDRs comprising the SEQ ID Nos disclosedherein with framework regions of the variable regions described hereinhaving an amino acid sequence that is at least 95%, 96%, 97% or 99%identical to the SEQ ID Nos disclosed herein.

In one embodiment, the anti-CD117 antibody, or antigen binding fragmentthereof, comprises a heavy chain variable region and a heavy chainconstant region having an amino acid sequence that is disclosed herein.In another embodiment, the anti-CD117 antibody, or antigen bindingfragment thereof, comprises a light chain variable region and a lightchain constant region having an amino acid sequence that is disclosedherein. In yet another embodiment, the anti-CD117 antibody, or antigenbinding fragment thereof, comprises a heavy chain variable region, alight chain variable region, a heavy chain constant region and a lightchain constant region having an amino acid sequence that is disclosedherein.

Methods of Identifying Anti-CD117 Antibodies

Methods for high throughput screening of antibody, or antibody fragment,libraries for molecules capable of binding CD117 (e.g., GNNK+ CD117) canbe used to identify and affinity mature antibodies useful for treatingcancers, autoimmune diseases, and conditioning a patient (e.g., a humanpatient) in need of hematopoietic stem cell therapy as described herein.Such methods include in vitro display techniques known in the art, suchas phage display, bacterial display, yeast display, mammalian celldisplay, ribosome display, mRNA display, and cDNA display, among others.The use of phage display to isolate ligands that bind biologicallyrelevant molecules has been reviewed, for example, in Felici et al.,Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys.Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology4:1-20, 1998, the disclosures of each of which are incorporated hereinby reference as they pertain to in vitro display techniques. Randomizedcombinatorial peptide libraries have been constructed to select forpolypeptides that bind cell surface antigens as described in Kay,Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol.Divers. 1:139-140, 1996, the disclosures of each of which areincorporated herein by reference as they pertain to the discovery ofantigen-binding molecules. Proteins, such as multimeric proteins, havebeen successfully phage-displayed as functional molecules (see, forexample, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell andMcCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each ofwhich are incorporated herein by reference as they pertain to the use ofin vitro display techniques for the discovery of antigen-bindingmolecules). In addition, functional antibody fragments, such as Fab andscFv fragments, have been expressed in in vitro display formats (see,for example, McCafferty et al., Nature 348:552-554, 1990; Barbas et al.,Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al.,Nature 352:624-628, 1991, the disclosures of each of which areincorporated herein by reference as they pertain to in vitro displayplatforms for the discovery of antigen-binding molecules). Thesetechniques, among others, can be used to identify and improve theaffinity of antibodies that bind CD117 (e.g., GNNK+ CD117) that can inturn be used to deplete endogenous hematopoietic stem cells in a patient(e.g., a human patient) in need of hematopoietic stem cell transplanttherapy.

In addition to in vitro display techniques, computational modelingtechniques can be used to design and identify antibodies, or antibodyfragments, in silico that bind CD117 (e.g., GNNK+ CD117). For example,using computational modeling techniques, one of skill in the art canscreen libraries of antibodies, or antibody fragments, in silico formolecules capable of binding specific epitopes, such as extracellularepitopes of this antigen. The antibodies, or antigen-binding fragmentsthereof, identified by these computational techniques can be used inconjunction with the therapeutic methods described herein, such as thecancer and autoimmune disease treatment methods described herein and thepatient conditioning procedures described herein.

Additional techniques can be used to identify antibodies, orantigen-binding fragments thereof, that bind CD117 (e.g., GNNK+ CD117)on the surface of a cell (e.g., a cancer cell, autoimmune cell, orhematopoietic stem cell) and that are internalized by the cell, forinstance, by receptor-mediated endocytosis. For example, the in vitrodisplay techniques described above can be adapted to screen forantibodies, or antigen-binding fragments thereof, that bind CD117 (e.g.,GNNK+ CD117) on the surface of a cancer cell, autoimmune cell, orhematopoietic stem cell and that are subsequently internalized. Phagedisplay represents one such technique that can be used in conjunctionwith this screening paradigm. To identify antibodies, or fragmentsthereof, that bind CD117 (e.g., GNNK+ CD117) and are subsequentlyinternalized by cancer cells, autoimmune cells, or hematopoietic stemcells, one of skill in the art can adapt the phage display techniquesdescribed, for example, in Williams et al., Leukemia 19:1432-1438, 2005,the disclosure of which is incorporated herein by reference in itsentirety. For example, using mutagenesis methods known in the art,recombinant phage libraries can be produced that encode antibodies,antibody fragments, such as scFv fragments, Fab fragments, diabodies,triabodies, and ¹⁰Fn3 domains, among others, that contain randomizedamino acid cassettes (e.g., in one or more, or all, of the CDRs orequivalent regions thereof or an antibody or antibody fragment). Theframework regions, hinge, Fc domain, and other regions of the antibodiesor antibody fragments may be designed such that they are non-immunogenicin humans, for instance, by virtue of having human germline antibodysequences or sequences that exhibit only minor variations relative tohuman germline antibodies.

Using phage display techniques described herein or known in the art,phage libraries containing randomized antibodies, or antibody fragments,covalently bound to the phage particles can be incubated with CD117(e.g., GNNK+ CD117) antigen, for instance, by first incubating the phagelibrary with blocking agents (such as, for instance, milk protein,bovine serum albumin, and/or IgG so as to remove phage encodingantibodies, or fragments thereof, that exhibit non-specific proteinbinding and phage that encode antibodies or fragments thereof that bindFc domains, and then incubating the phage library with a population ofhematopoietic stem cells. The phage library can be incubated with thetarget cells, such as cancer cells, autoimmune cells, or hematopoieticstem cells for a time sufficient to allow CD117-specific antibodies, orantigen-binding fragments thereof, (e.g., GNNK+ CD117-specificantibodies, or antigen-binding fragments thereof) to bind cell-surfaceCD117 (e.g., sell-surface GNNK+ CD117) antigen and to subsequently beinternalized by the cancer cells, autoimmune cells, or hematopoieticstem cells (e.g., from 30 minutes to 6 hours at 4° C., such as 1 hour at4° C.). Phage containing antibodies, or fragments thereof, that do notexhibit sufficient affinity for one or more of these antigens so as topermit binding to, and internalization by, cancer cells, autoimmunecells, or hematopoietic stem cells can subsequently be removed bywashing the cells, for instance, with cold (4° C.) 0.1 M glycine bufferat pH 2.8. Phage bound to antibodies, or fragments thereof, or that havebeen internalized by the cancer cells, autoimmune cells, orhematopoietic stem cells can be identified, for instance, by lysing thecells and recovering internalized phage from the cell culture medium.The phage can then be amplified in bacterial cells, for example, byincubating bacterial cells with recovered phage in 2×YT medium usingmethods known in the art. Phage recovered from this medium can then becharacterized, for instance, by determining the nucleic acid sequence ofthe gene(s) encoding the antibodies, or fragments thereof, insertedwithin the phage genome. The encoded antibodies, or fragments thereof,or can subsequently be prepared de novo by chemical synthesis (forinstance, of antibody fragments, such as scFv fragments) or byrecombinant expression (for instance, of full-length antibodies).

The internalizing capacity of the prepared antibodies, or fragmentsthereof, can be assessed, for instance, using radionuclideinternalization assays known in the art. For example, antibodies, orfragments thereof, identified using in vitro display techniquesdescribed herein or known in the art can be functionalized byincorporation of a radioactive isotope, such as ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I,¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, ⁶⁷Ga, ¹¹¹In, ⁹⁹Tc, ¹⁶⁹Yb, ¹⁸⁶Re, ⁶⁴Cu,⁶⁷Cu, ¹⁷⁷Lu, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ²¹²Bi, ²¹³Bi, or ²²⁵Ac. Forinstance, radioactive halogens, such as ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I, ¹²³I,¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, can be incorporated into antibodies, orfragments thereof, using beads, such as polystyrene beads, containingelectrophilic halogen reagents (e.g., Iodination Beads, Thermo FisherScientific, Inc., Cambridge, Mass.). Radiolabeled antibodies, orfragments thereof, can be incubated with cancer cells, autoimmune cells,or hematopoietic stem cells for a time sufficient to permitinternalization (e.g., from 30 minutes to 6 hours at 4° C., such as 1hour at 4° C.). The cells can then be washed to remove non-internalizedantibodies, or fragments thereof, (e.g., using cold (4° C.) 0.1 Mglycine buffer at pH 2.8). Internalized antibodies, or fragmentsthereof, can be identified by detecting the emitted radiation (e.g.,γ-radiation) of the resulting cancer cells, autoimmune cells, orhematopoietic stem cells in comparison with the emitted radiation (e.g.,γ-radiation) of the recovered wash buffer.

Antibody Drug Conjugates (ADCs)

Cytotoxins

Anti-CD117 antibodies, and antigen-binding fragments thereof, describedherein can be conjugated (linked) to a cytotoxin. In some embodiments,the cytotoxic molecule is conjugated to a cell internalizing antibody,or antigen-binding fragment thereof as disclosed herein such thatfollowing the cellular uptake of the antibody, or fragment thereof, thecytotoxin may access its intracellular target and mediate hematopoieticcell death. Any number of cytotoxins can be conjugated to the anti-CD117antibody, e.g., 1, 2, 3, 4, 5, 6, 7, or 8.

Cytotoxins suitable for use with the compositions and methods describedherein include DNA-intercalating agents, (e.g., anthracyclines), agentscapable of disrupting the mitotic spindle apparatus (e.g., vincaalkaloids, maytansine, maytansinoids, and derivatives thereof), RNApolymerase inhibitors (e.g., an amatoxin, such as α-amanitin, andderivatives thereof), and agents capable of disrupting proteinbiosynthesis (e.g., agents that exhibit rRNA N-glycosidase activity,such as saporin and ricin A-chain), among others known in the art.

In some embodiments, the cytotoxin is a microtubule-binding agent (forinstance, maytansine or a maytansinoid), an amatoxin, pseudomonasexotoxin A, deBouganin, diphtheria toxin, saporin, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof, or another cytotoxic compound described herein or known in theart.

Additional cytotoxins suitable for use with the compositions and methodsdescribed herein include, without limitation, 5-ethynyluracil,abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin,altretamine, ambamustine, amidox, amifostine, aminolevulinic acid,amrubicin, amsacrine, anagrelide, anastrozole, andrographolide,angiogenesis inhibitors, antarelix, anti-dorsalizing morphogeneticprotein-1, antiandrogen, prostatic carcinoma, antiestrogen,antineoplaston, antisense oligonucleotides, aphidicolin glycinate,apoptosis gene modulators, apoptosis regulators, apurinic acid,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin IIIderivatives, balanol, batimastat, BCR/ABL antagonists, benzochlorins,benzoylstaurosporine, beta lactam derivatives, beta-alethine,betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide,bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin,breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane,buthionine sulfoximine, calcipotriol, calphostin C, camptothecinderivatives (e.g., 10-hydroxy-camptothecin), capecitabine,carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, caseinkinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene and analogues thereof, clotrimazole, collismycin A,collismycin B, combretastatin A4, combretastatin analogues, conagenin,crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives,curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,dehydrodidemnin B, 2′deoxycoformycin (DCF), deslorelin, dexifosfamide,dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin,diphenyl spiromustine, discodermolide, docosanol, dolasetron,doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen,ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur,epothilones, epithilones, epristeride, estramustine and analoguesthereof, etoposide, etoposide 4′-phosphate (also referred to asetopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim,finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin,fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine,ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors,hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid,idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones,imiquimod, immunostimulant peptides, iobenguane, iododoxorubicin,ipomeanol, irinotecan, iroplact, irsogladine, isobengazole,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol,lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin,lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors,menogaril, rnerbarone, meterelin, methioninase, metoclopramide, MIFinhibitor, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone,mitolactol, mitomycin and analogues thereof, mitonafide, mitoxantrone,mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline,N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide,nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron,oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and analoguesthereof, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosanpolysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide,phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin,porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed,rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol,saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosicacid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine,tegafur, temozolomide, teniposide, thaliblastine, thiocoraline,tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine,vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb, amongothers.

Anti-CD117 antibodies, and antigen-binding fragments thereof, describedherein can be conjugated to a cytotoxin that is a microtubule bindingagent. As used herein, the term “microtubule-binding agent” refers to acompound which acts by disrupting the microtubular network that isessential for mitotic and interphase cellular function in a cell.Examples of microtubule-binding agents include, but are not limited to,maytasine, maytansinoids, and derivatives thereof, such as thosedescribed herein or known in the art, vinca alkaloids, such asvinblastine, vinblastine sulfate, vincristine, vincristine sulfate,vindesine, and vinorelbine, taxanes, such as docetaxel and paclitaxel,macrolides, such as discodermolides, cochicine, and epothilones, andderivatives thereof, such as epothilone B or a derivative thereof.

Maytansinoids

In some embodiments, the microtubule binding agent is a maytansine, amaytansinoid or a maytansinoid analog. Maytansinoids are mitototicinhibitors which bind microtubules and act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533. Maytansinoid drug moieties are attractive drug moieties inantibody drug conjugates because they are: (i) relatively accessible toprepare by fermentation or chemical modification, derivatization offermentation products, (ii) amenable to derivatization with functionalgroups suitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Examples of suitable maytansinoids include esters of maytansinol,synthetic maytansinol, and maytansinol analogs and derivatives. Includedherein are any cytotoxins that inhibit microtubule formation and thatare highly toxic to mammalian cells, as are maytansinoids, maytansinol,and maytansinol analogs, and derivatives.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,137,230;4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866;4,424,219; 4,450,254; 4,322,348; 4,362,663; 4,371,533; 5,208,020;5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; and7,473,796, the disclosures of each of which are incorporated herein byreference as they pertain to maytansinoids and derivatives thereof.

In some embodiments, the immunoconjugates of the invention utilize thethiol-containing maytansinoid (DM1), formally termedN²′-deacetyl-N²′-(3-mercapto-1-oxopropyl)-maytansine, as the cytotoxicagent. DM1 is represented by the structural formula (VII):

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoidN²′-deacetyl-N²′(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4)as the cytotoxic agent. DM4 is represented by the structural formula(V):

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN²′-deacetyl-N-²′(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the structural formula (VI):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of U.S. Pat. Nos. 5,208,020 and7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In some embodiments, the C-3 position servesas the position to chemically link the linking moiety, and in someparticular embodiments, the C-3 position of maytansinol serves as theposition to chemically link the linking moiety. There are many linkinggroups known in the art for making antibody-maytansinoid conjugates,including, for example, those disclosed in U.S. Pat. Nos. 5,208,020,6,441,163, and EP Patent No. 0425235 B1; Chari et al., Cancer Research52:127-131 (1992); and U.S. 2005/0169933 A1, the disclosures of whichare hereby expressly incorporated by reference. Additional linkinggroups are described and exemplified herein.

The present disclosure also includes various isomers and mixtures ofmaytansinoids and conjugates. Certain compounds and conjugates of thepresent invention may exist in various stereoisomeric, enantiomeric, anddiastereomeric forms. Several descriptions for producing suchantibody-maytansinoid conjugates are provided in U.S. Pat. Nos.5,208,020, 5,416,064 6,333,410, 6,441,163, 6,716,821, and 7,368,565,each of which is incorporated herein in its entirety.

A therapeutically effective number of maytansinoid molecules bound perantibody molecule can be determined by measuring spectrophotometricallythe ratio of the absorbance at 252 nm and 280 nm. An average of 3 to 4maytansinoid molecules conjugated per antibody molecule can enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although one molecule of toxin/antibodycan enhance cytotoxicity over antibody alone. The average number ofmaytansinoid molecules/antibody or antigen binding fragment thereof canbe, for example, 1-10 or 2-5.

Anthracyclines

In other embodiments, the antibodies and antigen-binding fragmentsthereof described herein can be conjugated to a cytotoxin that is ananthracycline molecule. Anthracyclines are antibiotic compounds thatexhibit cytotoxic activity. Studies have indicated that anthracyclinesmay operate to kill cells by a number of different mechanismsincluding: 1) intercalation of the drug molecules into the DNA of thecell thereby inhibiting DNA-dependent nucleic acid synthesis; 2)production by the drug of free radicals which then react with cellularmacromolecules to cause damage to the cells or 3) interactions of thedrug molecules with the cell membrane [see, e.g., C. Peterson et al.,”Transport And Storage Of Anthracycline In Experimental Systems And HumanLeukemia” in Anthracycline Antibiotics In Cancer Therapy; N. R. Bachur,“Free Radical Damage” id. at pp. 97-102]. Because of their cytotoxicpotential anthracyclines have been used in the treatment of numerouscancers such as leukemia, breast carcinoma, lung carcinoma, ovarianadenocarcinoma and sarcomas [see e.g., P. H-Wiernik, in Anthracycline:Current Status And New Developments p 11]. Commonly used anthracyclinesinclude doxorubicin, epirubicin, idarubicin and daunomycin.

The anthracycline analog, doxorubicin (ADRIAMYCINO) is thought tointeract with DNA by intercalation and inhibition of the progression ofthe enzyme topoisomerase II, which unwinds DNA for transcription.Doxorubicin stabilizes the topoisomerase II complex after it has brokenthe DNA chain for replication, preventing the DNA double helix frombeing resealed and thereby stopping the process of replication.Doxorubicin and daunorubicin (DAUNOMYCIN) are prototype cytotoxicnatural product anthracycline chemotherapeutics (Sessa et al., (2007)Cardiovasc. Toxicol. 7:75-79).

Pyrrolobenzodiazepines (PBDs)

In other embodiments, the anti-CD117 antibodies or antigen-bindingfragments thereof described herein can be conjugated to a cytotoxin thatis a pyrrolobenzodiazepine (PBD) or a cytotoxin that comprises a PBD.PBDs are natural products produced by certain actinomycetes and havebeen shown to be sequence selective DNA alkylating compounds. PBDcytotoxins include, but are not limited to, anthramycin, dimeric PBDs,and those disclosed in, for example, Hartley, J. A. (2011). “Thedevelopment of pyrrolobenzodiazepines as antitumour agents.” pert Opin.Inv. Drug, 20(6), 733-744; and Antonow, D.; Thurston, D. E. (2011)“Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines(PBDs).” Chem. Rev. 111: 2815-2864.

Calicheamicin

In other embodiments, the antibodies and antigen-binding fragmentsthereof described herein can be conjugated to a cytotoxin that is acalicheamicin molecule. The calicheamicin family of antibiotics arecapable of producing double-stranded DNA breaks at sub-picomolarconcentrations. For the preparation of conjugates of the calicheamicinfamily, see U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285;5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all to American CyanamidCompany). Structural analogues of calicheamicin which may be usedinclude, but are not limited to, those disclosed in, for example, Hinmanet al., Cancer Research 53:3336-3342 (1993), Lode et al., CancerResearch 58:2925-2928 (1998), and the aforementioned U.S. patents toAmerican Cyanamid.

Auristatins

Anti-CD117 antibodies, and antigen-binding fragments thereof, describedherein can be conjugated to a cytotoxin that is an auristatin (U.S. Pat.Nos. 5,635,483; 5,780,588). Auristatins are anti-mitotic agents thatinterfere with microtubule dynamics, GTP hydrolysis, and nuclear andcellular division (Woyke et al (2001) Antimicrob. Agents and Chemother.45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) andantifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.42:2961-2965). (U.S. Pat. Nos. 5,635,483; 5,780,588). The auristatindrug moiety may be attached to the antibody through the N-(amino)terminus or the C-(carboxyl) terminus of the peptidic drug moiety (WO02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in Senter et al,Proceedings of the American Association for Cancer Research, Volume 45,Abstract Number 623, presented Mar. 28, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE, wherein the wavy lineindicates the point of covalent attachment to the linker of anantibody-linker conjugate (-L-Z-Ab, as described herein).

Another exemplary auristatin embodiment is MMAF, wherein the wavy lineindicates the point of covalent attachment to the linker of anantibody-linker conjugate (-L-Z-Ab, as described herein), as disclosedin US 2005/0238649:

Auristatins may be prepared according to the methods of: U.S. Pat. Nos.5,635,483; 5,780,588; Pettit et al (1989) J. Am. Chem. Soc.111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277;Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J.Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

Amatoxins

In some embodiments, the cytotoxin of the antibody-drug conjugate is anRNA polymerase inhibitor. In some embodiments, the RNA polymeraseinhibitor is an amatoxin or derivative thereof.

In some embodiments, the cytotoxin is an amatoxin or derivative thereof,such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin,amaninamide, amanullin, amanullinic acid, and proamanullin. Structuresof the various naturally occurring amatoxins are represented by formulaIII, and are disclosed in, e.g., Zanotti et al., Int. J. Peptide ProteinRes. 30, 1987, 450-459.

In one embodiment, the cytotoxin is an amanitin. For instance, theantibodies, or antigen-binding fragments, described herein may be boundto an amatoxin so as to form a conjugate represented by the formulaAb-Z-L-Am, wherein Ab is the antibody, or antigen-binding fragmentthereof, L is a linker, Z is a chemical moiety and Am is an amatoxin.Many positions on amatoxins or derivatives thereof can serve as theposition to covalently bond the linking moiety L, and, hence theantibodies or antigen-binding fragments thereof. In some embodiments,Am-L-Z is represented by formula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene,

a dipeptide, —(C═O)—, a peptide, or a combination thereof; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117).

In some embodiments, Am contains exactly one R_(C) substituent.

In some embodiments, the linker comprises a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker includes—((CH₂)_(n) where n is 6. In some embodiments, L-Z is

where S is a sulfur atom which represents the reactive substituentpresent within an antibody, or antigen-binding fragment thereof, thatbinds CD117 (e.g., from the —SH group of a cysteine residue).

In some embodiments, L-Z is

In some embodiments, Am-L-Z-Ab is one of:

wherein X is —S—, —S(O)—, or —SO₂—.

In some embodiments, Am-L-Z-Ab is

In some embodiments, Am-L-Z-Ab is

In some embodiments, Am-L-Z-Ab is

In some embodiments, Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(C);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117); and

wherein Am contains exactly one R_(C) substituent. In some embodiments,the linker includes —((CH₂)_(n) where n is 6. In some embodiments, L-Zis

In some embodiments, L-Z is

In some embodiments, Am-L-Z is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene,

a dipeptide, —(C═O)—, a peptide, or a combination thereof;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, or antigen-binding fragment thereof, that bindsCD117 (such as GNNK+ CD117); and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

In some embodiments, L-Z is

In some embodiments, R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form to form a 5-memberedheterocycloalkyl group of formula:

wherein Y is —(C═O)—, —(C═S)—, —(C═NR_(E))—, or —(CR_(E)R_(E))—; and

R_(E) and R_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form:

R₃ is H or R_(C);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH;

X is —S—, —S(O)—, or —SO₂—; and

wherein R_(C) and R_(D) are each as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form:

R₃ is H or R_(C);

R₄ and R₅ are each independently H, OH, OR_(C), R_(C), or OR_(D);

R₆ and R₇ are each H;

R₅ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH;

X is —S—, —S(O)—, or —SO₂—; and

wherein R_(C) is as defined above.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₅ is OH or NH₂;

R₉ is H or OH;

X is —S—, —S(O)—, or —SO₂—; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2016/0002298, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2014/0294865, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H, OH, OR_(C), or R_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am-L-Z is represented by formula (IA) or formula(IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681, aswell as in US 2016/0089450, the disclosures of each of which areincorporated herein by reference in their entirety.

Additional amatoxins that may be used for conjugation to an antibody, orantigen-binding fragment thereof, in accordance with the compositionsand methods described herein are described, for example, in WO2016/142049; WO 2016/071856; WO 2017/046658; and WO2018/115466, thedisclosures of each of which are incorporated herein by reference intheir entirety.

In some embodiments, Am-L-Z is represented by formula (II), formula IIA,or formula IIB

wherein X is S, SO, or SO₂; R₁ is H or a linker covalently bound to theantibody or antigen-binding fragment thereof through a chemical moietyZ, formed from a coupling reaction between a reactive substituentpresent on the linker and a reactive substituent present within anantibody, or antigen-binding fragment thereof; and R₂ is H or a linkercovalently bound to the antibody or antigen-binding fragment thereofthrough a chemical moiety Z, formed from a coupling reaction between areactive substituent present on the linker and a reactive substituentpresent within an antibody, or antigen-binding fragment thereof; whereinwhen R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is the linker.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, R₁ is the linker and R₂ is H,and the linker and chemical moiety, together as L-Z, is

In some embodiments, Am-L-Z-Ab is:

In some embodiments, Am-L-Z-Ab is:

In some embodiments, the cytotoxin is an α-amanitin. In someembodiments, the α-amanitin is a compound of formula III. In someembodiments, the α-amanitin of formula III is attached to an anti-CD117antibody via a linker L. The linker L may be attached to the α-amanitinof formula III at any one of several possible positions (e.g., any ofR¹-R⁹) to provide an α-amanitin-linker conjugate of formula I, IA, IB,II, IIA, IIB, IV, IVA or IVB. In some embodiments, the linker isattached at position R¹. In some embodiments, the linker is attached atposition R². In some embodiments, the linker is attached at position R³.In some embodiments, the linker is attached at position R⁴. In someembodiments, the linker is attached at position R⁵. In some embodiments,the linker is attached at position R⁶. In some embodiments, the linkeris attached at position R⁷. In some embodiments, the linker is attachedat position R⁸. In some embodiments, the linker is attached at positionR⁹. In some embodiments, the linker includes a hydrazine, a disulfide, athioether or a dipeptide. In some embodiments, the linker includes adipeptide selected from Val-Ala and Val-Cit. In some embodiments, thelinker includes a para-aminobenzyl group (PAB). In some embodiments, thelinker includes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is a β-amanitin. In some embodiments,the β-amanitin is a compound of formula I. In some embodiments, theα-amanitin is a compound of formula III. In some embodiments, theβ-amanitin of formula III is attached to an anti-CD117 antibody via alinker L. The linker L may be attached to the β-amanitin of formula IIIat any one of several possible positions (e.g., any of R¹-R⁹) to providean β-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. Insome embodiments, the linker is attached at position R¹. In someembodiments, the linker is attached at position R². In some embodiments,the linker is attached at position R³. In some embodiments, the linkeris attached at position R⁴. In some embodiments, the linker is attachedat position R⁵. In some embodiments, the linker is attached at positionR⁶. In some embodiments, the linker is attached at position R⁷. In someembodiments, the linker is attached at position R⁸. In some embodiments,the linker is attached at position R⁹. In some embodiments, the linkerincludes a hydrazine, a disulfide, a thioether or a dipeptide. In someembodiments, the linker includes a dipeptide selected from Val-Ala andVal-Cit. In some embodiments, the linker includes a para-aminobenzylgroup (PAB). In some embodiments, the linker includes the moietyPAB-Cit-Val. In some embodiments, the linker includes the moietyPAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is a γ-amanitin. In some embodiments,the γ-amanitin is a compound of formula III. In some embodiments, theγ-amanitin of formula III is attached to an anti-CD117 antibody via alinker L. The linker L may be attached to the γ-amanitin of formula IIIat any one of several possible positions (e.g., any of R¹-R⁹) to providean γ-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. Insome embodiments, the linker is attached at position R¹. In someembodiments, the linker is attached at position R². In some embodiments,the linker is attached at position R³. In some embodiments, the linkeris attached at position R⁴. In some embodiments, the linker is attachedat position R⁵. In some embodiments, the linker is attached at positionR⁶. In some embodiments, the linker is attached at position R⁷. In someembodiments, the linker is attached at position R⁸. In some embodiments,the linker is attached at position R⁹. In some embodiments, the linkerincludes a hydrazine, a disulfide, a thioether or a dipeptide. In someembodiments, the linker includes a dipeptide selected from Val-Ala andVal-Cit. In some embodiments, the linker includes a para-aminobenzylgroup (PAB). In some embodiments, the linker includes the moietyPAB-Cit-Val. In some embodiments, the linker includes the moietyPAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linkeris—PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L andthe chemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is a ε-amanitin. In some embodiments,the ε-amanitin is a compound of formula III. In some embodiments, theε-amanitin of formula III is attached to an anti-CD117 antibody via alinker L. The linker L may be attached to the ε-amanitin of formula IIIat any one of several possible positions (e.g., any of R¹-R⁹) to providean ε-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. Insome embodiments, the linker is attached at position R¹. In someembodiments, the linker is attached at position R². In some embodiments,the linker is attached at position R³. In some embodiments, the linkeris attached at position R⁴. In some embodiments, the linker is attachedat position R⁵. In some embodiments, the linker is attached at positionR⁶. In some embodiments, the linker is attached at position R⁷. In someembodiments, the linker is attached at position R⁸. In some embodiments,the linker is attached at position R⁹. In some embodiments, the linkerincludes a hydrazine, a disulfide, a thioether or a dipeptide. In someembodiments, the linker includes a dipeptide selected from Val-Ala andVal-Cit. In some embodiments, the linker includes a para-aminobenzylgroup (PAB). In some embodiments, the linker includes the moietyPAB-Cit-Val. In some embodiments, the linker includes the moietyPAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is an amanin. In some embodiments,the amanin is a compound of formula III. In some embodiments, the amaninof formula III is attached to an anti-CD117 antibody via a linker L. Thelinker L may be attached to the amanin of formula III at any one ofseveral possible positions (e.g., any of R¹-R⁹) to provide anamanin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In someembodiments, the linker is attached at position R¹. In some embodiments,the linker is attached at position R². In some embodiments, the linkeris attached at position R³. In some embodiments, the linker is attachedat position R⁴. In some embodiments, the linker is attached at positionR⁵. In some embodiments, the linker is attached at position R⁶. In someembodiments, the linker is attached at position R⁷. In some embodiments,the linker is attached at position R⁸. In some embodiments, the linkeris attached at position R⁹. In some embodiments, the linker includes ahydrazine, a disulfide, a thioether or a dipeptide. In some embodiments,the linker includes a dipeptide selected from Val-Ala and Val-Cit. Insome embodiments, the linker includes a para-aminobenzyl group (PAB). Insome embodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is an amaninamide. In someembodiments, the amaninamide is a compound of formula III. In someembodiments, the amaninamide of formula III is attached to an anti-CD117antibody via a linker L. The linker L may be attached to the amaninamideof formula III at any one of several possible positions (e.g., any ofR¹-R⁹) to provide an amaninamide-linker conjugate of formula I, IA, IB,II, IIA, or IIB. In some embodiments, the linker is attached at positionR¹. In some embodiments, the linker is attached at position R². In someembodiments, the linker is attached at position R³. In some embodiments,the linker is attached at position R⁴. In some embodiments, the linkeris attached at position R⁵. In some embodiments, the linker is attachedat position R⁶. In some embodiments, the linker is attached at positionR⁷. In some embodiments, the linker is attached at position R⁸. In someembodiments, the linker is attached at position R⁹. In some embodiments,the linker includes a hydrazine, a disulfide, a thioether or adipeptide. In some embodiments, the linker includes a dipeptide selectedfrom Val-Ala and Val-Cit. In some embodiments, the linker includes apara-aminobenzyl group (PAB). In some embodiments, the linker includesthe moiety PAB-Cit-Val. In some embodiments, the linker includes themoiety PAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is an amanullin. In some embodiments,the amanullin is a compound of formula III. In some embodiments, theamanullin of formula III is attached to an anti-CD117 antibody via alinker L. The linker L may be attached to the amanullin of formula IIIat any one of several possible positions (e.g., any of R¹-R⁹) to providean amanullin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. Insome embodiments, the linker is attached at position R¹. In someembodiments, the linker is attached at position R². In some embodiments,the linker is attached at position R³. In some embodiments, the linkeris attached at position R⁴. In some embodiments, the linker is attachedat position R⁵. In some embodiments, the linker is attached at positionR⁶. In some embodiments, the linker is attached at position R⁷. In someembodiments, the linker is attached at position R⁸. In some embodiments,the linker is attached at position R⁹. In some embodiments, the linkerincludes a hydrazine, a disulfide, a thioether or a dipeptide. In someembodiments, the linker includes a dipeptide selected from Val-Ala andVal-Cit. In some embodiments, the linker includes a para-aminobenzylgroup (PAB). In some embodiments, the linker includes the moietyPAB-Cit-Val. In some embodiments, the linker includes the moietyPAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. I

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is an amanullinic acid. In someembodiments, the amanullinic acid is a compound of formula III. In someembodiments, the amanullinic acid of formula III is attached to ananti-CD117 antibody via a linker L. The linker L may be attached to theamanullinic acid of formula III at any one of several possible positions(e.g., any of R¹-R⁹) to provide an amanullinic acid-linker conjugate offormula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker isattached at position R¹. In some embodiments, the linker is attached atposition R². In some embodiments, the linker is attached at position R³.In some embodiments, the linker is attached at position R⁴. In someembodiments, the linker is attached at position R⁵. In some embodiments,the linker is attached at position R⁶. In some embodiments, the linkeris attached at position R⁷. In some embodiments, the linker is attachedat position R⁸. In some embodiments, the linker is attached at positionR⁹. In some embodiments, the linker includes a hydrazine, a disulfide, athioether or a dipeptide. In some embodiments, the linker includes adipeptide selected from Val-Ala and Val-Cit. In some embodiments, thelinker includes a para-aminobenzyl group (PAB). In some embodiments, thelinker includes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

In some embodiments, the cytotoxin is a proamanullin. In someembodiments, the proamanullin is a compound of formula III. In someembodiments, the proamanullin of formula III is attached to ananti-CD117 antibody via a linker L. The linker L may be attached to theproamanullin of formula III at any one of several possible positions(e.g., any of R¹-R⁹) to provide an proamanullin-linker conjugate offormula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker isattached at position R¹. In some embodiments, the linker is attached atposition R². In some embodiments, the linker is attached at position R³.In some embodiments, the linker is attached at position R⁴. In someembodiments, the linker is attached at position R⁵. In some embodiments,the linker is attached at position R⁶. In some embodiments, the linkeris attached at position R⁷. In some embodiments, the linker is attachedat position R⁸. In some embodiments, the linker is attached at positionR⁹. In some embodiments, the linker includes a hydrazine, a disulfide, athioether or a dipeptide. In some embodiments, the linker includes adipeptide selected from Val-Ala and Val-Cit. In some embodiments, thelinker includes a para-aminobenzyl group (PAB). In some embodiments, thelinker includes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker L and thechemical moiety Z, taken together as L-Z, is

Synthetic methods of making amatoxin are described in U.S. Pat. No.9,676,702, which is incorporated by reference herein.

Antibodies, or antigen-binding fragments, for use with the compositionsand methods described herein can be conjugated to an amatoxin, such asα-amanitin or a variant thereof, using conjugation techniques known inthe art or described herein. For instance, antibodies, orantigen-binding fragments thereof, that recognize and bind CD117 (suchas GNNK+ CD117) can be conjugated to an amatoxin, such as α-amanitin ora variant thereof, as described in US 2015/0218220, the disclosure ofwhich is incorporated herein by reference as it pertains, for example,to amatoxins, such as α-amanitin and variants thereof, as well ascovalent linkers that can be used for covalent conjugation.

Exemplary antibody-drug conjugates useful in conjunction with themethods described herein may be formed by the reaction of an antibody,or antigen-binding fragment thereof, with an amatoxin that is conjugatedto a linker containing a substituent suitable for reaction with areactive residue on the antibody, or antigen-binding fragment thereof.Amatoxins that are conjugated to a linker containing a substituentsuitable for reaction with a reactive residue on the antibody, orantigen-binding fragment thereof, described herein include, withoutlimitation, 7′C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(maleimido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxin;7′C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;(R)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;(S)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1yl)methyl)-amatoxin;7′C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7′C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxin;7′C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaeicosyl)piperidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7′C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-(pyridine-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;6′O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin;6′O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxin;6′O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxin;6′O-((6-(maleimido)hexyl)carbamoyl)-amatoxin;6′O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxin;6′O-(6-(2-bromoacetamido)hexyl)-amatoxin;7′C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;6′O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6′O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;6′O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6′O-((6-aminooxy)hexyl)-amatoxin; and6′O-(6-(2-iodoacetamido)hexyl)-amatoxin. The foregoing linkers, amongothers useful in conjunction with the compositions and methods describedherein, are described, for example, in US Patent Application PublicationNo. 2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

Additional cytotoxins that can be conjugated to antibodies, orantigen-binding fragments thereof, that recognize and bind CD117 (suchas GNNK+ CD117 for use in directly treating a cancer, automminecondition, or for conditioning a patient (e.g., a human patient) inpreparation for hematopoietic stem cell transplant therapy include,without limitation, 5-ethynyluracil, abiraterone, acylfulvene,adecypenol, adozelesin, aldesleukin, altretamine, ambamustine, amidox,amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide,anastrozole, andrographolide, angiogenesis inhibitors, antarelix,anti-dorsalizing morphogenetic protein-1, antiandrogen, prostaticcarcinoma, antiestrogen, antineoplaston, antisense oligonucleotides,aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators,apurinic acid, asulacrine, atamestane, atrimustine, axinastatin 1,axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatinIII derivatives, balanol, batimastat, BCR/ABL antagonists,benzochlorins, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitors,bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistrateneA, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine,budotitane, buthionine sulfoximine, calcipotriol, calphostin C,camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine,carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, caseinkinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene and analogues thereof, clotrimazole, collismycin A,collismycin B, combretastatin A4, combretastatin analogues, conagenin,crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives,curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,dehydrodidemnin B, 2′deoxycoformycin (DCF), deslorelin, dexifosfamide,dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin,diphenyl spiromustine, discodermolide, docosanol, dolasetron,doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen,ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur,epothilones, epithilones, epristeride, estramustine and analoguesthereof, etoposide, etoposide 4′-phosphate (also referred to asetopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim,finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin,fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine,ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors,hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid,idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones,imiquimod, immunostimulant peptides, iobenguane, iododoxorubicin,ipomeanol, irinotecan, iroplact, irsogladine, isobengazole,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol,lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin,lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors,menogaril, rnerbarone, meterelin, methioninase, metoclopramide, MIFinhibitor, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone,mitolactol, mitomycin and analogues thereof, mitonafide, mitoxantrone,mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline,N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide,nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron,oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and analoguesthereof, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosanpolysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide,phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin,porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed,rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol,saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosicacid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine,tegafur, temozolomide, teniposide, thaliblastine, thiocoraline,tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine,vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb, amongothers.

Linkers for Chemical Conjugation

A variety of linkers can be used to conjugate antibodies, orantigen-binding fragments, as described herein (e.g., antibodies, orantigen-binding fragments thereof, that recognize and bind CD117 (suchas GNNK+ CD117) with a cytotoxic molecule.

The term “Linker” as used herein means a divalent chemical moietycomprising a covalent bond or a chain of atoms that covalently attachesan antibody or fragment thereof (Ab) to a drug moiety (D) to formantibody-drug conjugates of the present disclosure (ADCs; Ab-Z-L-D,where D is a cytotoxin). Suitable linkers have two reactive termini, onefor conjugation to an antibody and the other for conjugation to acytotoxin. The antibody conjugation reactive terminus of the linker(reactive moiety, Z) is typically a site that is capable of conjugationto the antibody through a cysteine thiol or lysine amine group on theantibody, and so is typically a thiol-reactive group such as a doublebond (as in maleimide) or a leaving group such as a chloro, bromo, iodo,or an R-sulfanyl group, or an amine-reactive group such as a carboxylgroup; while the antibody conjugation reactive terminus of the linker istypically a site that is capable of conjugation to the cytotoxin throughformation of an amide bond with a basic amine or carboxyl group on thecytotoxin, and so is typically a carboxyl or basic amine group. When theterm “linker” is used in describing the linker in conjugated form, oneor both of the reactive termini will be absent (such as reactive moietyZ, having been converted to chemical moiety Z) or incomplete (such asbeing only the carbonyl of the carboxylic acid) because of the formationof the bonds between the linker and/or the cytotoxin, and between thelinker and/or the antibody or antigen-binding fragment thereof. Suchconjugation reactions are described further herein below.

In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the drug unit fromthe antibody in the intracellular environment. In yet other embodiments,the linker unit is not cleavable and the drug is released, for example,by antibody degradation. The linkers useful for the present ADCs arepreferably stable extracellularly, prevent aggregation of ADC moleculesand keep the ADC freely soluble in aqueous media and in a monomericstate. Before transport or delivery into a cell, the ADC is preferablystable and remains intact, i.e. the antibody remains linked to the drugmoiety. The linkers are stable outside the target cell and may becleaved at some efficacious rate inside the cell. An effective linkerwill: (i) maintain the specific binding properties of the antibody; (ii)allow intracellular delivery of the conjugate or drug moiety; (iii)remain stable and intact, i.e. not cleaved, until the conjugate has beendelivered or transported to its targeted site; and (iv) maintain acytotoxic, cell-killing effect or a cytostatic effect of the cytotoxicmoiety. Stability of the ADC may be measured by standard analyticaltechniques such as mass spectroscopy, HPLC, and the separation/analysistechnique LC/MS. Covalent attachment of the antibody and the drug moietyrequires the linker to have two reactive functional groups, i.e.bivalency in a reactive sense. Bivalent linker reagents which are usefulto attach two or more functional or biologically active moieties, suchas peptides, nucleic acids, drugs, toxins, antibodies, haptens, andreporter groups are known, and methods have been described theirresulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques;Academic Press: New York, p. 234-242).

Linkers include those that may be cleaved, for instance, by enzymatichydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysisunder basic conditions, oxidation, disulfide reduction, nucleophiliccleavage, or organometallic cleavage (see, for example, Leriche et al.,Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which isincorporated herein by reference as it pertains to linkers suitable forcovalent conjugation).

Linkers hydrolyzable under acidic conditions include, for example,hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides,orthoesters, acetals, ketals, or the like. (See, e.g., U.S. Pat. Nos.5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661, the disclosure of each of which is incorporated hereinby reference in its entirety as it pertains to linkers suitable forcovalent conjugation. Such linkers are relatively stable under neutralpH conditions, such as those in the blood, but are unstable at below pH5.5 or 5.0, the approximate pH of the lysosome.

Linkers cleavable under reducing conditions include, for example, adisulfide. A variety of disulfide linkers are known in the art,including, for example, those that can be formed using SATA(N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931;Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935, the disclosure of each of whichis incorporated herein by reference in its entirety as it pertains tolinkers suitable for covalent conjugation.

Additional linkers suitable for the synthesis of drug-antibodyconjugates conjugates as described herein include those capable ofreleasing a cytotoxin by a 1,6-elimination process (a “self-immolative”group), such as p-aminobenzyl alcohol (PABC), p-aminobenzyl (PAB),6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagentsdescribed in Jain et al., Pharm. Res. 32:3526-3540, 2015, the disclosureof which is incorporated herein by reference in its entirety.

In some embodiments, the linker includes a self-immolative group such asthe aforementioned PAB or PABC (para-aminobenzyloxycarbonyl), which aredisclosed in, for example, Carl et al., J. Med. Chem. (1981) 24:479-480;Chakravarty et al (1983) J. Med. Chem. 26:638-644; U.S. Pat. No.6,214,345; US20030130189; US20030096743; U.S. Pat. No. 6,759,509;US20040052793; U.S. Pat. Nos. 6,218,519; 6,835,807; 6,268,488;US20040018194; WO98/13059; US20040052793; U.S. Pat. Nos. 6,677,435;5,621,002; US20040121940; WO2004/032828). Other such chemical moietiescapable of this process (“self-immolative linkers”) include methylenecarbamates and heteroaryl groups such as aminothiazoles,aminoimidazoles, aminopyrimidines, and the like. Linkers containing suchheterocyclic self-immolative groups are disclosed in, for example, U.S.Patent Publication Nos. 20160303254 and 20150079114, and U.S. Pat. No.7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US2005/0256030; de Groot et al (2001) J. Org. Chem. 66:8815-8830; and U.S.Pat. No. 7,223,837.

Linkers susceptible to enzymatic hydrolysis can be, e.g., apeptide-containing linker that is cleaved by an intracellular peptidaseor protease enzyme, including, but not limited to, a lysosomal orendosomal protease. One advantage of using intracellular proteolyticrelease of the therapeutic agent is that the agent is typicallyattenuated when conjugated and the serum stabilities of the conjugatesare typically high. In some embodiments, the peptidyl linker is at leasttwo amino acids long or at least three amino acids long. Exemplary aminoacid linkers include a dipeptide, a tripeptide, a tetrapeptide or apentapeptide. Examples of suitable peptides include those containingamino acids such as Valine, Alanine, Citrulline (Cit), Phenylalanine,Lysine, Leucine, and Glycine. Amino acid residues which comprise anamino acid linker component include those occurring naturally, as wellas minor amino acids and non-naturally occurring amino acid analogs,such as citrulline. Exemplary dipeptides include valine-citrulline (vcor val-cit) and alanine-phenylalanine (af or ala-phe). Exemplarytripeptides include glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). In some embodiments, the linkerincludes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys,Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkerscontaining dipeptides such as Val-Cit or Phe-Lys are disclosed in, forexample, U.S. Pat. No. 6,214,345, the disclosure of which isincorporated herein by reference in its entirety as it pertains tolinkers suitable for covalent conjugation. In some embodiments, thelinker includes a dipeptide selected from Val-Ala and Val-Cit. In someembodiments, a dipeptide is used in combination with a self-immolativelinker.

Linkers suitable for use herein further may include one or more groupsselected from C₁-C₆ alkylene, C₁-C₆ heteroalkylene, C₂-C₆ alkenylene,C₂-C₆ heteroalkenylene, C₂-C₆alkynylene, C₂-C₆ heteroalkynylene,C₃-C₆cycloalkylene, heterocycloalkylene, arylene, heteroarylene, andcombinations thereof, each of which may be optionally substituted.Non-limiting examples of such groups include (CH₂)_(n), (CH₂CH₂O)_(n),and —(C═O)(CH₂)_(n)— units, wherein n is an integer from 1-6,independently selected for each occasion.

In some embodiments, the linker may include one or more of a hydrazine,a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, aheterocyclic self-immolative group, an optionally substitutedC₁-C₆alkyl, an optionally substituted C₁-C₆ heteroalkyl, an optionallysubstituted C₂-C₆ alkenyl, an optionally substituted C₂-C₆heteroalkenyl, an optionally substituted C₂-C₆ alkynyl, an optionallysubstituted C₂-C₆ heteroalkynyl, an optionally substitutedC₃-C₆cycloalkyl, an optionally substituted heterocycloalkyl, anoptionally substituted aryl, an optionally substituted heteroaryl, acyl,—(C═O)—, or —(CH₂CH₂O)_(n)— group, wherein n is an integer from 1-6. Oneof skill in the art will recognize that one or more of the groups listedmay be present in the form of a bivalent (diradical) species, e.g.,C₁-C₆ alkylene and the like.

In some embodiments, the linker includes a p-aminobenzyl group (PAB). Inone embodiment, the p-aminobenzyl group is disposed between thecytotoxic drug and a protease cleavage site in the linker. In oneembodiment, the p-aminobenzyl group is part of ap-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzylgroup is part of a p-aminobenzylamido unit.

In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

In some embodiments, the linker comprises a combination of one or moreof a peptide, oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, PAB,Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

In some embodiments, the linker comprises a —(C═O)(CH₂)_(n)— unit,wherein n is an integer from 1-6.

In some embodiments, the linker comprises a —(CH₂)_(n)— unit, wherein nis an integer from 2 to 6.

Linkers that can be used to conjugate an antibody, or antigen-bindingfragment thereof, to a cytotoxic agent include those that are covalentlybound to the cytotoxic agent on one end of the linker and, on the otherend of the linker, contain a chemical moiety formed from a couplingreaction between a reactive substituent present on the linker and areactive substituent present within the antibody, or antigen-bindingfragment thereof, that binds CD117 (such as GNNK+ CD117). Reactivesubstituents that may be present within an antibody, or antigen-bindingfragment thereof, that binds CD117 (such as GNNK+ CD117) include,without limitation, hydroxyl moieties of serine, threonine, and tyrosineresidues; amino moieties of lysine residues; carboxyl moieties ofaspartic acid and glutamic acid residues; and thiol moieties of cysteineresidues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl),haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkylmoieties of non-naturally occurring amino acids.

Examples of linkers useful for the synthesis of drug-antibody conjugatesconjugates include those that contain electrophiles, such as Michaelacceptors (e.g., maleimides), activated esters, electron-deficientcarbonyl compounds, and aldehydes, among others, suitable for reactionwith nucleophilic substituents present within antibodies orantigen-binding fragments, such as amine and thiol moieties. Forinstance, linkers suitable for the synthesis of drug-antibody conjugatesinclude, without limitation, succinimidyl4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyliodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidylester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among othersdescribed, for instance, Liu et al., 18:690-697, 1979, the disclosure ofwhich is incorporated herein by reference as it pertains to linkers forchemical conjugation. Additional linkers include the non-cleavablemaleimidocaproyl linkers, which are particularly useful for theconjugation of microtubule-disrupting agents such as auristatins, aredescribed by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, thedisclosure of which is incorporated herein by reference as it pertainsto linkers for chemical conjugation.

It will be recognized by one of skill in the art that any one or more ofthe chemical groups, moieties and features disclosed herein may becombined in multiple ways to form linkers useful for conjugation of theantibodies and cytotoxins as disclosed herein. Further linkers useful inconjunction with the compositions and methods described herein, aredescribed, for example, in U.S. Patent Application Publication No.2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

Linkers useful in conjunction with the antibody-drug described hereininclude, without limitation, linkers containing chemical moieties formedby coupling reactions as depicted in Table 3, below. Curved linesdesignate points of attachment to the antibody, or antigen-bindingfragment, and the cytotoxic molecule, respectively.

TABLE 3 Exemplary chemical moieties Z formed by coupling reactions inthe formation of antibody-drug Exemplary Coupling Reactions ChemicalMoiety Z Formed by Coupling Reactions [3 + 2] Cycloaddition

[3 + 2] Cycloaddition

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition

Michael addition

Michael addition

Imine condensation, Amidation

Imine condensation

Disulfide formation

Thiol alkylation

Condensation, Michael addition

One of skill in the art will recognize that a reactive substituent Zattached to the linker and a reactive substituent on the antibody orantigen-binding fragment thereof, are engaged in the covalent couplingreaction to produce the chemical moiety Z, and will recognize thereactive substituent Z. Therefore, antibody-drug conjugates useful inconjunction with the methods described herein may be formed by thereaction of an antibody, or antigen-binding fragment thereof, with alinker or cytotoxin-linker conjugate, as described herein, the linker orcytotoxin-linker conjugate including a reactive substituent Z, suitablefor reaction with a reactive substituent on the antibody, orantigen-binding fragment thereof, to form the chemical moiety Z.

As depicted in Table 3, examples of suitably reactive substituents onthe linker and antibody or antigen-binding fragment thereof include anucleophile/electrophile pair (e.g., a thiol/haloalkyl pair, anamine/carbonyl pair, or a thiol/α,β-unsaturated carbonyl pair, and thelike), a diene/dienophile pair (e.g., an azide/alkyne pair, or adiene/α,β-unsaturated carbonyl pair, among others), and the like.Coupling reactions between the reactive substitutents to form thechemical moiety Z include, without limitation, thiol alkylation,hydroxyl alkylation, amine alkylation, amine or hydroxylaminecondensation, hydrazine formation, amidation, esterification, disulfideformation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2]Huisgen cycloaddition, among others), nucleophilic aromaticsubstitution, electrophilic aromatic substitution, and other reactivemodalities known in the art or described herein. Preferably, the linkercontains an electrophilic functional group for reaction with anucleophilic functional group on the antibody, or antigen-bindingfragment thereof.

Reactive substituents that may be present within an antibody, orantigen-binding fragment thereof, as disclosed herein include, withoutlimitation, nucleophilic groups such as (i) N-terminal amine groups,(ii) side chain amine groups, e.g. lysine, (iii) side chain thiolgroups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where theantibody is glycosylated. Reactive substituents that may be presentwithin an antibody, or antigen-binding fragment thereof, as disclosedherein include, without limitation, hydroxyl moieties of serine,threonine, and tyrosine residues; amino moieties of lysine residues;carboxyl moieties of aspartic acid and glutamic acid residues; and thiolmoieties of cysteine residues, as well as propargyl, azido, haloaryl(e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl,and haloheteroalkyl moieties of non-naturally occurring amino acids. Insome embodiments, the reactive substituents present within an antibody,or antigen-binding fragment thereof as disclosed herein include, areamine or thiol moieties. Certain antibodies have reducible interchaindisulfides, i.e. cysteine bridges. Antibodies may be made reactive forconjugation with linker reagents by treatment with a reducing agent suchas DTT (dithiothreitol). Each cysteine bridge will thus form,theoretically, two reactive thiol nucleophiles. Additional nucleophilicgroups can be introduced into antibodies through the reaction of lysineswith 2-iminothiolane (Traut's reagent) resulting in conversion of anamine into a thiol. Reactive thiol groups may be introduced into theantibody (or fragment thereof) by introducing one, two, three, four, ormore cysteine residues (e.g., preparing mutant antibodies comprising oneor more non-native cysteine amino acid residues). U.S. Pat. No.7,521,541 teaches engineering antibodies by introduction of reactivecysteine amino acids.

In some embodiments, the reactive moiety Z attached to the linker is anucleophilic group which is reactive with an electrophilic group presenton an antibody. Useful electrophilic groups on an antibody include, butare not limited to, aldehyde and ketone carbonyl groups. The heteroatomof a nucleophilic group can react with an electrophilic group on anantibody and form a covalent bond to the antibody. Useful nucleophilicgroups include, but are not limited to, hydrazide, oxime, amino,hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide.

In some embodiments, Z is the product of a reaction between reactivenucleophilic substituents present within the antibodies, orantigen-binding fragments thereof, such as amine and thiol moieties, anda reactive electrophilic substituent Z. For instance, Z may be a Michaelacceptor (e.g., maleimide), activated ester, electron-deficient carbonylcompound, or an aldehyde, among others.

In some embodiments, the ADC comprises an anti-CD117 antibody conjugatedto an amatoxin of any of formulae I, IA, IB, II, IIA, or IIB asdisclosed herein via a linker and a chemical moiety Z. In someembodiments, the linker includes a hydrazine, a disulfide, a thioetheror a dipeptide. In some embodiments, the linker includes a dipeptideselected from Val-Ala and Val-Cit. In some embodiments, the linkerincludes a para-aminobenzyl group (PAB). In some embodiments, the linkerincludes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. Insome embodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the linker includes a —(CH₂)_(n)— unit, where n isan integer from 2-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is—(CH₂)_(n)—. In some embodiments, the linker is —((CH₂)_(n)—, wherein nis 6.

In some embodiments, the chemical moiety Z is selected from Table 3. Insome embodiments, the chemical moiety Z is

where S is a sulfur atom which represents the reactive substituentpresent within an antibody, or antigen-binding fragment thereof, thatbinds CD117 (e.g., from the —SH group of a cysteine residue).

In some embodiments, the linker L and the chemical moiety Z, takentogether as L-Z, is

One of skill in the art will recognize the linker-reactive substituentgroup structure, prior to conjugation with the antibody or antigenbinding fragment thereof, includes a maleimide as the group Z. Theforegoing linker moieties and amatoxin-linker conjugates, among othersuseful in conjunction with the compositions and methods describedherein, are described, for example, in U.S. Patent ApplicationPublication No. 2015/0218220 and Patent Application Publication No.WO2017/149077, the disclosure of each of which is incorporated herein byreference in its entirety.

Preparation of Antibody-Drug Conjugates

In the ADCs of formula I as disclosed herein, an antibody or antigenbinding fragment thereof is conjugated to one or more cytotoxic drugmoieties (D), e.g. about 1 to about 20 drug moieties per antibody,through a linker L and a chemical moiety Z as disclosed herein. The ADCsof the present disclosure may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a reactive substituent ofan antibody or antigen binding fragment thereof with a bivalent linkerreagent to form Ab-Z-L as described herein above, followed by reactionwith a drug moiety D; or (2) reaction of a reactive substituent of adrug moiety with a bivalent linker reagent to form D-L-Z, followed byreaction with a reactive substituent of an antibody or antigen bindingfragment thereof as described herein above to form an ADC of formulaD-L-Z-Ab, such as Am-Z-L-Ab. Additional methods for preparing ADC aredescribed herein.

In another aspect, the antibody or antigen binding fragment thereof hasone or more lysine residues that can be chemically modified to introduceone or more sulfhydryl groups. The ADC is then formed by conjugationthrough the sulfhydryl group's sulfur atom as described herein above.The reagents that can be used to modify lysine include, but are notlimited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another aspect, the antibody or antigen binding fragment thereof canhave one or more carbohydrate groups that can be chemically modified tohave one or more sulfhydryl groups. The ADC is then formed byconjugation through the sulfhydryl group's sulfur atom as describedherein above.

In yet another aspect, the antibody can have one or more carbohydrategroups that can be oxidized to provide an aldehyde (—CHO) group (see,for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The ADCis then formed by conjugation through the corresponding aldehyde asdescribed herein above. Other protocols for the modification of proteinsfor the attachment or association of cytotoxins are described in Coliganet al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons(2002), incorporated herein by reference.

Methods for the conjugation of linker-drug moieties to cell-targetedproteins such as antibodies, immunoglobulins or fragments thereof arefound, for example, in U.S. Pat. Nos. 5,208,020; 6,441,163;WO2005037992; WO2005081711; and WO2006/034488, all of which are herebyexpressly incorporated by reference in their entirety.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

Methods of Treatment

As described herein, hematopoietic stem cell transplant therapy can beadministered to a subject in need of treatment so as to populate orre-populate one or more blood cell types. Hematopoietic stem cellsgenerally exhibit multi-potency, and can thus differentiate intomultiple different blood lineages including, but not limited to,granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets),monocytes (e.g., monocytes, macrophages), dendritic cells, microglia,osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells).Hematopoietic stem cells are additionally capable of self-renewal, andcan thus give rise to daughter cells that have equivalent potential asthe mother cell, and also feature the capacity to be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis.

Hematopoietic stem cells can thus be administered to a patient defectiveor deficient in one or more cell types of the hematopoietic lineage inorder to re-constitute the defective or deficient population of cells invivo, thereby treating the pathology associated with the defect ordepletion in the endogenous blood cell population. The compositions andmethods described herein can thus be used to treat a non-malignanthemoglobinopathy (e.g., a hemoglobinopathy selected from the groupconsisting of sickle cell anemia, thalassemia, Fanconi anemia, aplasticanemia, and Wiskott-Aldrich syndrome). Additionally or alternatively,the compositions and methods described herein can be used to treat animmunodeficiency, such as a congenital immunodeficiency. Additionally oralternatively, the compositions and methods described herein can be usedto treat an acquired immunodeficiency (e.g., an acquiredimmunodeficiency selected from the group consisting of HIV and AIDS).The compositions and methods described herein can be used to treat ametabolic disorder (e.g., a metabolic disorder selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromaticleukodystrophy).

Additionally or alternatively, the compositions and methods describedherein can be used to treat a malignancy or proliferative disorder, suchas a hematologic cancer, myeloproliferative disease. In the case ofcancer treatment, the compositions and methods described herein may beadministered to a patient so as to deplete a population of endogenoushematopoietic stem cells prior to hematopoietic stem celltransplantation therapy, in which case the transplanted cells can hometo a niche created by the endogenous cell depletion step and establishproductive hematopoiesis. This, in turn, can re-constitute a populationof cells depleted during cancer cell eradication, such as duringsystemic chemotherapy. Exemplary hematological cancers that can betreated using the compositions and methods described herein include,without limitation, acute myeloid leukemia, acute lymphoid leukemia,chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma,diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, as well asother cancerous conditions, including neuroblastoma.

Additional diseases that can be treated with the compositions andmethods described herein include, without limitation, adenosinedeaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditarylymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storagediseases, thalassemia major, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

The antibodies, antigen-binding fragments thereof, and conjugatesdescribed herein may be used to induce solid organ transplant tolerance.For instance, the compositions and methods described herein may be usedto deplete or ablate a population of cells from a target tissue (e.g.,to deplete hematopoietic stem cells from the bone marrow stem cellniche). Following such depletion of cells from the target tissues, apopulation of stem or progenitor cells from an organ donor (e.g.,hematopoietic stem cells from the organ donor) may be administered tothe transplant recipient, and following the engraftment of such stem orprogenitor cells, a temporary or stable mixed chimerism may be achieved,thereby enabling long-term transplant organ tolerance without the needfor further immunosuppressive agents. For example, the compositions andmethods described herein may be used to induce transplant tolerance in asolid organ transplant recipient (e.g., a kidney transplant, lungtransplant, liver transplant, and heart transplant, among others). Thecompositions and methods described herein are well-suited for use inconnection the induction of solid organ transplant tolerance, forinstance, because a low percentage temporary or stable donor engraftmentis sufficient to induce long-term tolerance of the transplanted organ.

In addition, the compositions and methods described herein can be usedto treat cancers directly, such as cancers characterized by cells thatare CD117+. For instance, the compositions and methods described hereincan be used to treat leukemia, particularly in patients that exhibitCD117+ leukemic cells. By depleting CD117+ cancerous cells, such asleukemic cells, the compositions and methods described herein can beused to treat various cancers directly. Exemplary cancers that may betreated in this fashion include hematological cancers, such as acutemyeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia,chronic lymphoid leukemia, multiple myeloma, diffuse large B-celllymphoma, and non-Hodgkin's lymphoma.

Acute myeloid leukemia (AML) is a cancer of the myeloid line of bloodcells, characterized by the rapid growth of abnormal white blood cellsthat build up in the bone marrow and interfere with the production ofnormal blood cells. AML is the most common acute leukemia affectingadults, and its incidence increases with age. The symptoms of AML arecaused by replacement of normal bone marrow with leukemic cells, whichcauses a drop in red blood cells, platelets, and normal white bloodcells. As an acute leukemia, AML progresses rapidly and may be fatalwithin weeks or months if left untreated. In one embodiment, theanti-CD117 ADCs described herein are used to treat AML in a humanpatient in need thereof. In certain embodiments the anti-CD117 ADCtreatment depletes AML cells in the treated subjects. In someembodiments 50% or more of the AML cells are depleted. In otherembodiments, 60% or more of the AML cells are depleted, or 70% or moreof the AML cells are depleted, or 80% of more or 90% or more, or 95% ormore of the AML cells are depleted. In certain embodiments theanti-CD117 ADC treatments is a single dose treatment. In certainembodiments the single dose anti-CD117 ADC treatment depletes 60%, 70%,80%, 90% or 95% or more of the AML cells.

In addition, the compositions and methods described herein can be usedto treat autoimmune disorders. For instance, an antibody, orantigen-binding fragment thereof, can be administered to a subject, suchas a human patient suffering from an autoimmune disorder, so as to killa CD117+ immune cell. The CD117+ immune cell may be an autoreactivelymphocyte, such as a T-cell that expresses a T-cell receptor thatspecifically binds, and mounts an immune response against, a selfantigen. By depleting self-reactive, CD117+ cells, the compositions andmethods described herein can be used to treat autoimmune pathologies,such as those described below. Additionally or alternatively, thecompositions and methods described herein can be used to treat anautoimmune disease by depleting a population of endogenous hematopoieticstem cells prior to hematopoietic stem cell transplantation therapy, inwhich case the transplanted cells can home to a niche created by theendogenous cell depletion step and establish productive hematopoiesis.This, in turn, can re-constitute a population of cells depleted duringautoimmune cell eradication.

Autoimmune diseases that can be treated using the compositions andmethods described herein include, without limitation, psoriasis,psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes),rheumatoid arthritis (RA), human systemic lupus (SLE), multiplesclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis,acute disseminated encephalomyelitis (ADEM), Addison's disease, alopeciauniversalis, ankylosing spondylitisis, antiphospholipid antibodysyndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balodisease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas'disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Crohn's disease, cicatricalpemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinindisease, CREST syndrome, Degos disease, discoid lupus, dysautonomia,endometriosis, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease,Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, Hidradenitissuppurativa, idiopathic and/or acute thrombocytopenic purpura,idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis,juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease,Meniere disease, mixed connective tissue disease (MCTD), myastheniagravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), opticneuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,polychondritis, polymyositis and dermatomyositis, primary biliarycirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgiarheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter'ssyndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome,stiff person syndrome, Takayasu's arteritis, temporal arteritis (alsoknown as “giant cell arteritis”), ulcerative colitis, collagenouscolitis, uveitis, vasculitis, vitiligo, vulvodynia (“vulvarvestibulitis”), and Wegener's granulomatosis.

Routes of Administration and Dosing

ADCs, antibodies, or antigen-binding fragments thereof, or describedherein can be administered to a patient (e.g., a human patient sufferingfrom cancer, an autoimmune disease, or in need of hematopoietic stemcell transplant therapy) in a variety of dosage forms. For instance,antibodies, or antigen-binding fragments thereof, described herein canbe administered to a patient suffering from cancer, an autoimmunedisease, or in need of hematopoietic stem cell transplant therapy in theform of an aqueous solution, such as an aqueous solution containing oneor more pharmaceutically acceptable excipients. Pharmaceuticallyacceptable excipients for use with the compositions and methodsdescribed herein include viscosity-modifying agents. The aqueoussolution may be sterilized using techniques known in the art.

Pharmaceutical formulations comprising anti-CD117 ADCs or antibodies asdescribed herein are prepared by mixing such ADC or anti-CD117 antibodywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

The ADCs, antibodies, or antigen-binding fragments, described herein maybe administered by a variety of routes, such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intraocularly, or parenterally. The most suitable route foradministration in any given case will depend on the particular antibody,or antigen-binding fragment, administered, the patient, pharmaceuticalformulation methods, administration methods (e.g., administration timeand administration route), the patient's age, body weight, sex, severityof the diseases being treated, the patient's diet, and the patient'sexcretion rate.

The effective dose of an ADC, antibody, or antigen-binding fragmentthereof, described herein can range, for example from about 0.001 toabout 100 mg/kg of body weight per single (e.g., bolus) administration,multiple administrations, or continuous administration, or to achieve anoptimal serum concentration (e.g., a serum concentration of 0.0001-5000μg/mL) of the antibody, antigen-binding fragment thereof. The dose maybe administered one or more times (e.g., 2-10 times) per day, week, ormonth to a subject (e.g., a human) suffering from cancer, an autoimmunedisease, or undergoing conditioning therapy in preparation for receiptof a hematopoietic stem cell transplant. In the case of a conditioningprocedure prior to hematopoietic stem cell transplantation, the ADC,antibody, or antigen-binding fragment thereof, can be administered tothe patient at a time that optimally promotes engraftment of theexogenous hematopoietic stem cells, for instance, from 1 hour to 1 week(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7days) or more prior to administration of the exogenous hematopoieticstem cell transplant.

EXAMPLES

The data provided in FIGS. 1, 22, and 23 disclosed herein represent databased on the compositions and methods described herein, including thesequences disclosed in Tables 1 and 2, and are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention.

In Examples 1 through 3 (including the data provided in FIGS. 2-4),CD117-ADC is a CK6, a fully human antagonistic antibody specific forCD117 that is conjugated to a toxin capable of depleting cycling andnon-cycling cells. The isotype control ADC is a non-targeted monoclonalhuman IgG antibody conjugated to the same toxin as CD117-ADC. In theexperiments described in Examples 1 to 3, the control naked CD117antibody is the same CK6 antibody used in the CD117-ADC.

Example 1 In Vitro Analysis of an Anti-CD117-ADC Using an In Vitro CellKilling Assay

The anti-CD117 antibody used in the ADC in the following Example isanti-CD117 antibody CK6 conjugated to amatoxin. For in vitro killingassays using Kasumi-1 cells, Kasumi-1 cells were grown according to ATCCguidelines. More specifically, Kasumi-1 cells were cultured for threedays in the presence of CD117-ADC or the controls. Cell viability wasmeasured by Celltiter Glo. For in vitro killing assays using Human HSCs(i.e., isolated primary human CD34+ selected Bone Marrow Cells (BMCs)),human CD34+ BMCs were cultured for five days with CD117-ADC or thecontrols in the presence of IL-6, TPO, and FLT-3 ligand, with or withoutSCF. Live cell counts were determined by flow cytometry. Killing oftotal nucleated cells in culture mirrored the results shown in FIG. 4.

The results in FIGS. 2A and 2B indicate that CD117-ADC is highlyeffective at killing CD117 expressing cell lines (e.g., Kasumi-1 cells)or primary human CD34⁺ cells in vitro, demonstrating greater than 90%killing of the leukemia cell line Kasumi-1 (FIG. 2A; IC₅₀=1.6 pM), andequally effective killing of primary human CD34+ bone marrow cellsduring in vitro culture (FIG. 2B; IC₅₀=21 pM). Thus, CD117-ADC is highlyeffective at killing CD117 expressing cell lines and primary human CD34+cells.

Example 2 In Vivo HSC Depletion Assay Using an Anti-CD117-ADC

The anti-CD117 antibody used in the ADC in the following Example isantibody CK6 conjugated to an amatoxin. In vivo HSC depletion assayswere conducted using humanized mice (purchased from JacksonLaboratories). As schematically shown in FIG. 3A, CD117-ADC (singleinjection of 0.3 mg/kg CD117-ADC) or controls were administered in asingle dose on day 0 to the humanized NSG mice. PBMCs were collected ondays 7, 14, and 21 and examined by flow cytometry. On day 21 (postinjection), the presence or absence human CD34+ cells in the bone marrowwas quantitated. The percent of human myeloid (FIG. 3B) or thepercentage of T cells (FIG. 3C) present in the peripheral blood ofCD117-ADC or control treated mice (non-CD117 binding isotype matchedantibody), were determined and expressed as a percent of that cellpopulation prior to treatment (normalized to baseline). The absolutenumber of CD34+ cells in the bone marrow of CD117-ADC or control treatedmice 21 days after a single administration of the antibody drugconjugate are shown in FIG. 3D.

The results indicate that humanized NSG mice treated with CD117-ADC hadgreater than 90% depletion of human HSPCs in the bone marrow, 21 daysfollowing a single administration of the ADC. Similar results wereobtained for CD34+ CD90+ and CD34+, CD90+, CD117+ cells (data notshown). The specificity of CD117-ADC for HSPCs was confirmed by thepresence of stable peripheral human lymphocyte populations, and theabsence of human myeloid cells, indicating a lack of stem and progenitorcells capable of replenishing these short-lived cells. Accordingly,these data indicate that the CD117-ADC depletes human CD34+ cells in thebone marrow of humanized NSG mice.

Example 3 In Vivo Tumor Study Using an Anti-CD117-ADC

The anti-CD117 antibody used in the ADC and in the naked antibody usedin the following ample is CK6. The drug conjugated to the CK6 antibodyin this Example is an amatoxin. 5 million Kasumi-1 cells were IVinjected into naïve NSG mice. On day 7 (FIG. 4C) or on day 42 (FIG. 4D)mice were treated with CD117-ADC (0.3 mg/kg) or controls (either 0.3mg/kg or 1.0 mg/kg for CD117-antibody group). The bone marrow of micewas analyzed for the presence of Kasumi-1 cells following euthanasia.FIG. 4A depicts the phenotypic analysis of Kasumi-1 cells in culture.FIG. 4B depicts the phenotypic analysis of Kasumi-1 cells from bonemarrow of tumor bearing mice at time of euthanasia. FIG. 4C graphicallydepicts the survival curve of mice treated with CD117-ADC or controls.FIG. 4D graphically depicts the survival curve of mice (with a greatertumor burden than those treated in FIG. 4C) treated with CD117-ADC orcontrols. There were at least 5 mice in the untreated, CD117-antibody,and CD117-ADC groups.

As demonstrated in FIG. 4C, the animals treated with a single dose ofthe anti-CD117-ADC show 100% survival, even at 150 days post-injection,while the animals administered the controls show significant decreasesin percent survival at 110 days post-injection. As demonstrated in FIG.4D, the animals treated with a single dose of the anti-CD117-ADC show100% survival, even at 130 days post-injection, while the animalsadministered the controls show significant decreases in percent survivalat 100 days post-injection. These results also indicate that theCD117-ADC was well tolerated at the doses given and enabled completesurvival of the treated mice.

Example 4 In Vivo HSC Depletion/Engraftment Assays Using anAnti-CD117-ADC

The anti-CD117 antibody used in the ADC in the following example is 2B8.

In vivo HSC depletion assays were conducted using B6 mice (purchasedfrom Jackson Laboratories). As schematically shown in FIG. 5A,anti-CD117-ADC (single injection of 1.0 mg/kg i.v. anti-CD117-ADC) orcontrols were administered in a single dose on day 0 to the B6 mice. 4days following the single administration of the anti-CD117 ADC, 1×10⁷donor CD45.1+ donor cells were infused. Blood was collected on weeks 4,8, 16, 20 and bone marrow was collected on week 20 and examined by flowcytometry. Post-transplant, the presence or absence Kit+SCA+CD150+CD48+cells in the bone marrow was quantitated from the B6 mice treated withanti-CD117-ADC or a control (FIG. 5B). The percentage of donor chimerismin the B6 mice treated with anti-CD117-ADC or a control are shown inFIG. 5C.

The results indicate that B6 mice treated with anti-CD117-ADC (i.e.,CD117-Saporin) had greater than 95% depletion of Kit+SCA+CD150+CD48+cells, following a single administration of the ADC. Further, theresults indicate that B6 mice treated with anti-CD117-ADC (i.e.,CD117-Saporin) had greater than 70% donor chimerism post treatment,indicating that treatment with an anti-CD117-ADC (i.e., CD117-Saporin)enables robust donor engraftment.

Example 5 Analysis of Non-Human Primate Pharmacokinetics

A non-human primate pharmacokinetic assay was performed to determine thechange in the concentration (ng/mL) of a isotype control antibody (i.e.,“wild type antibody”) compared to an Fc-modified CK6 variant antibody(i.e., an H435A Fc mutation; variant refers to the Fc modification) topossess a shorter half life as a function time (post-administration).The results in FIG. 6 demonstrate that the CK6 variant antibody ischaracterized by a significantly shorter half-life in a non-humanprimate.

Example 6 In Vivo HSC Depletion Assay Using an Anti-CD117-ADC

An experiment was performed to compare the Fc-modified CK6 variantantibody (i.e., an H435A Fc mutation; variant in this example refers tothe Fc modification) from Example 5 to a wild-type anti-CD117 ADC(containing the CK6 antibody conjugated to an amatoxin). In vivo HSCdepletion assays were conducted using humanized mice (purchased fromJackson Laboratories). The Fc-modified CK6 variant antibody (i.e., anH435A Fc mutation) from Example 5 was administered as a single injectionof 1 mg/kg CD117-ADC, 0.3 mg/kg CD117-ADC, 0.1 mg/kg CD117-ADC, or 0.03mg/kg CD117-ADC) to the humanized mouse model. In addition, a wild-typeanti-CD117 ADC was similarly administered as a single injection of 1mg/kg, 0.3 mg/kg, 0.1 mg/kg, or 0.03 mg/kg) to the humanized mice on day0. Bone marrow was collected on day 21 and examined by flow cytometry(FIG. 7A). The absolute number of CD34+ cells in the bone marrow oftreated or control treated mice on day 21 after a single administrationof the treatment regimen are shown in FIG. 7B.

The results indicate that humanized NSG mice treated with both theFc-modified CK6 variant antibody and the wild-type (unmodified Fcregion) anti-CD117 ADC showed significant depletion of human HSPCs inthe bone marrow, 21 days following a single administration of the ADCwhen compared to the controls (FIG. 7B). Further, flow cytometry data inFIG. 7A indicated that the wild-type anti-CD117-ADC significantlydepleted human CD34+ cells in the bone marrow of humanized NSG micecompared to controls (i.e., an isotype ADC (1 mg/kg) and an anti-CD117antibody (CK6) (1 mg/kg)).

Example 7 In Vivo HSC Depletion Assay Using an Anti-CD117-ADC

An additional experiment was performed to compare the Fc-modified CK6variant antibody (i.e., an H435A Fc mutation; variant in this examplerefers to the Fc region) from Example 5 to a wild-type anti-CD117 ADC(CK6 antibody with an unsubstituted Fc region conjugated to anamatoxin). In vivo HSC depletion assays were conducted using humanizedmice (purchased from Jackson Laboratories). The Fc-modified CK6 variantantibody (i.e., an H435A Fc mutation) from Example 5 was administered asa single injection of 1 mg/kg CD117-ADC, 0.3 mg/kg CD117-ADC, 0.1 mg/kgCD117-ADC, or 0.03 mg/kg CD117-ADC) to the humanized mouse model. Inaddition, a wild-type anti-CD117 ADC was similarly administered as asingle injection of 1 mg/kg CD117-ADC, 0.3 mg/kg CD117-ADC, 0.1 mg/kgCD117-ADC, or 0.03 mg/kg CD117-ADC) to the humanized mice on day 0.Blood was collected on day 21 and examined by flow cytometry. Thepercentage of human CD33+ (FIG. 8A), human CD3+ (FIG. 8B), and humanCD19+ (FIG. 8C), of treated or control treated mice on day 21 relativeto baseline are shown.

The results indicate that humanized NSG mice treated with theFc-modified CK6 variant antibody and the wild-type anti-CD117 ADC showedsignificant depletion of human CD33+ myeloid cells (FIG. 8A) relative tobaseline, 21 days following a single administration of the treatmentregimen and indicate that both the anti-CD117 Fc-modified CK6 ADC andthe wild-type anti-CD117 CK6 ADC depleted myeloid cells as a result ofthe depletion of early progenitor cells. Further, the results indicatethat both the anti-CD117 Fc-modified CK6 ADC and the wild-typeanti-CD117 CK6 ADC do not significantly deplete human peripherallymphocytes, e.g., T cells (modeled as human CD3+ cells) and B cells(modeled as CD19+ cells) 21 days following a single administration ofthe treatment regimen.

Example 8 Identification of Novel Anti-CD117 Antibodies

A human Fab phage display library was created based on a derivative ofthe human CK6 antibody (i.e., HC-1/LC-1 (Ab1) in order to identifyimproved anti-CD117 antibodies that had better affinity properties thanCK6 while maintaining the functional antagonistic and internalizingcharacteristics of CK6. The CK6 derivative used as the bases for thescreen was Ab1, which is a variant of CK6 containing conservative aminoacid substitutions within the light chain and heavy chain variableregions. Once the library was established, the screening process wasperformed according to standard phage display affinity maturationmethodology known in the art. Briefly, the HC-1 was combined with amixed donor-derived pool of human kappa light chains. Phage displayselections were subsequently performed to selectively identify cloneswith improved off-rates after iterative rounds of panning. Antibodieswere then screened to identify novel anti-CD117 antibodies with alteredaffinity to human CD117, e.g., an improved off rate of the antibodywhile maintaining kinetic characteristics of the CK6 antibody, includinginternalization.

To confirm binding to the desired target, purified antibodies wereanalyzed for binding to purified recombinant human CD117 ectodomain bybio-layer interferometry (BLI). Binding analysis of the antibodiesidentified from the phage display campaigns revealed a number ofderivatives with improved off-rate kinetics as compared to HC-1/LC-1(FIG. 1). The apparent kinetic values are provided in Table 4, whichlists the apparent monovalent affinity (K_(D)), apparent associationrate (k_(on)), and apparent dissociation rate (k_(dis)) of the indicatedpurified IgG to purified human CD117 ectodomain (R&D Systems #332-SR) asmeasured by BLI.

TABLE 4 K_(D)(M) k_(on) (1/Ms) K_(dis)(1/s) 001 (Ab1) 2.81E−09 3.02E+058.48E−04 002 (Ab2) 7.29E−10 3.70E+05 2.70E−04 003 (Ab3) 1.02E−093.46E+05 3.52E−04 004 (Ab4) 1.18E−09 3.45E+05 4.05E−04 005 (Ab5)1.21E−09 3.80E+05 4.61E−04 006 (Ab6) 3.72E−09 3.15E+05 1.17E−03 007(Ab7) 9.90E−10 2.75E+05 2.72E−04 008 (Ab8) 9.08E−10 2.90E+05 2.64E−04009 (Ab9) 1.51E−09 2.83E+05 4.27E−04 010 (Ab10) 1.68E−09 3.48E+055.85E−04 011 (Ab11) 1.41E−09 3.36E+05 4.74E−04 012 (Ab12) 9.11E−102.94E+05 2.68E−04 013 (Ab13) 1.55E−09 3.14E+05 4.88E−04 014 (Ab14)2.03E−09 2.84E+05 5.77E−04 015 (Ab15) 9.79E−10 2.65E+05 2.60E−04 016(Ab16) 2.93E−09 3.24E+05 9.49E−04 017 (Ab17) 6.72E−10 4.56E+05 3.06E−04018 (Ab18) 2.22E−08 2.21E+05 4.91E−03 019 (Ab19) 2.35E−09 2.05E+054.81E−04 020 (Ab20) 2.54E−10 7.94E+05 2.02E−04 021 (Ab21) 7.15E−106.12E+05 4.37E−04 022 (Ab22) 9.64E−11 2.29E+06 2.21E−04 023 (Ab23)1.49E−09 4.78E+05 7.13E−04 024 (Ab24) 9.86E−10 1.11E+05 1.10E−04 025(Ab25) 5.20E−10 2.94E+06 1.53E−03 027 (Ab27) 5.30E−10 7.12E+05 3.78E−04028 (Ab28) 4.03E−10 9.14E+05 3.68E−04

A subset of these antibodies were engineered to look at combinations ofaffinity modifying substitutions that had not been sampled in the phagedisplay campaigns and may provide affinity benefit as cooperativesequence variations. Antibodies 77, 79, and 81 (Ab77, Ab79, and Ab81,respectively) representative examples of subsidiary derivatives and aredescribed in more detail below.

Antibody 77 (Having HC-77/LC-77)

The heavy chain variable region (VH) amino acid sequence of Antibody 77(Ab77) is provided below as SEQ ID NO: 7. The VH CDR amino acidsequences of Ab77 are underlined below and are as follows: TYWIG (VHCDR1; SEQ ID NO: 163); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); andHGRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3).

Ab77 VH sequence (SEQ ID NO: 7)QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS

The light chain variable region (VL) amino acid sequence of Ab77 isprovided below as SEQ ID NO 91. The VL CDR amino acid sequences of Ab77underlined below and are as follows: RASQGVISALA (VL CDR1; SEQ ID NO:164); DASILES (VL CDR2; SEQ ID NO: 165); and QQFNSYPLT (VL CDR3; SEQ IDNO: 166).

Ab77 VL sequence (SEQ ID NO: 91)DIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTKVEIKAntibody 79 (Having HC79/LC79)

The heavy chain variable region (VH) amino acid sequence of Ab79 isprovided below as SEQ ID NO: 7. The VH CDR amino acid sequences of Ab79are underlined below and are as follows: TYWIG (VH CDR1; SEQ ID NO:163); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGRGYNGYEGAFDI (VHCDR3; SEQ ID NO: 3).

Ab79 VH sequence (SEQ ID NO: 7)QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS

The light chain variable region (VL) amino acid sequence of Ab79 isprovided below as SEQ ID NO: 93. The VL CDR amino acid sequences of Ab79underlined below and are as follows: RASQGVGSALA (VL CDR1; SEQ ID NO:167); DASILES (VL CDR2; SEQ ID NO: 165); and QQFNSYPLT (VL CDR3; SEQ IDNO: 166).

Ab79 VL sequence (SEQ ID NO: 93)DIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWYQQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTKVEIKAntibody 81 (Having HC-81/LC-81)

The heavy chain variable region (VH) amino acid sequence of Ab81 isprovided below as SEQ ID NO: 7. The VH CDR amino acid sequences of Ab81underlined below and are as follows: TYWIG (VH CDR1; SEQ ID NO: 163);IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGRGYNGYEGAFDI (VH CDR3;SEQ ID NO: 3).

Ab81 VH sequence (SEQ ID NO: 7)QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSS

The light chain variable region (VL) amino acid sequence of Ab81 isprovided below as SEQ ID NO 95. The VL CDR amino acid sequences of Ab81underlined below and are as follows: RASQGVISALA (VL CDR1; SEQ ID NO:164); DASTLES (VL CDR2; SEQ ID NO: 168); and QQFNSYPLT (VL CDR3; SEQ IDNO: 166).

Ab81 VL sequence (SEQ ID NO: 95)DIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTKVEIK

This subset of clones consisting of library output iterations in theCDR1 and CDR2 of the light chain allowed for the analysis of cooperativesubstitutions in subsequent in vitro binding assays.

Example 9 In Vitro Antibody Binding Studies

Antibodies identified in Example 8 were tested for binding. Antibodybinding studies were performed at 25 degrees celsius in 1× PBSsupplemented with 0.1% w/v bovine serum albumin with a Pall ForteBioOctet Red96 using biolayer interferometry (BLI). The indicated purifiedhuman antibody (as an IgG1) was immobilized onto anti-human Fcbiosensors (AHC; Pall ForteBio 18-5063) and incubated with 33.3 nM and11 nM CD117 ectodomain (R&D Systems #332-SR)

The resulting binding intervals, which represented the association anddissociation curves were depicted in FIG. 9. The apparent monovalentaffinity (KD), apparent association rate (kon), and apparentdissociation rate (kdis) are determined by local full fitting with a 1:1binding mode as calculated by Fortebio data analysis software version 10of the indicated purified IgG (i.e., the HC-77/LC-106 IgG; HC-109/LC-110IgG; and HC-113/LC-114 IgG) to purified human CD117 ectodomain (R&DSystems #332-SR) were depicted in Table 5. Table 5 lists the apparentmonovalent affinity (K_(D)), apparent association rate (k_(on)), andapparent dissociation rate (k_(OFF)) of the indicated purified IgG topurified human CD117 ectodomain (R&D Systems #332-SR). The resultsdemonstrate a purified IgG (i.e., the HC-77/LC-77 IgG; HC-79/LC-79 IgG;and HC-81/LC-81 IgG) binds with high affinity to the purified humanCD117 ectodomain and is characterized by a significantly slowerkdis(1/s) when compared to the HC-1/LC-1 purified IgG.

TABLE 5 Antibody K_(D) (M) k_(on)(1/Ms) K_(dis)(1/s) HC-1/LC-1 (Ab1)3.10 × 10⁻⁹  4.20 × 10⁵ 1.30 × 10⁻³ (control) HC-77/LC-77 (Ab77) 6.84 ×10⁻¹⁰ 3.15 × 10⁵ 2.15 × 10⁻⁴ HC-79/LC-79 (Ab79) 1.52 × 10⁻⁹  3.16 × 10⁵4.78 × 10⁻⁴ HC-81/LC-81 (Ab 81) 8.77 × 10⁻¹⁰ 2.88 × 10⁵ 2.52 × 10⁻⁴

As described in Table 5, each of antibodies Ab77, Ab79, and Ab81 hadimproved binding (K_(D)) compared to parent antibody Ab1. Surprisingly,the high level of affinity was maintained, while improving thedissociation rate.

Example 10 Identification of Anti-CD117 Antibody 85 (Ab85), Anti-CD117Antibody 86 (Ab86), Anti-CD117 Antibody 87 (Ab87), Anti-CD117 Antibody88 (Ab88), and Anti-CD117 Antibody 89 (Ab89)

In addition to the screen described in Examples 8 and 9, a second screenbased on antibody CK6 was also performed. An scFv phage display librarywas created based on a derivative of the human CK6 antibody (as inExample 8 the CK6 variant was Ab1). Briefly, a small synthetic libraryof CDRH3 variants was generated to remove a potential deamidation site(NG) and was introduced into a large diversity library of either theIGHV5-51 or the IGHV1-46 human framework. This screen was a challengegiven the position of the amino acid in the CDR3 region of the heavychain. The synthetic library was combined with a large diversity libraryof the IGKV1-39 light chain human framework. Phage display selectionswere then performed to selectively identify clones with improvedoff-rates after iterative rounds of panning. Antibodies were thenscreened to identify novel anti-CD117 antibodies with improved affinityto human CD117. Certain antibodies, including the antibodies identifiedbelow, were identified using the screen.

Antibodies 85 (Ab85), 86 (Ab86), 87 (Ab87), 88 (Ab88), and 89 (Ab89)were identified in the screen as a novel therapeutic human anti-CD117antibody. The heavy chain and light chain variable regions of Ab85,Ab86, Ab87, Ab88, and Ab89 (including the CDR domains) are describedbelow in Table 6.

TABLE 6 Antibody name Backbone Amino acid sequence HC-85 hIgG1EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 143)LC-85 hKappa DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQANGFPLTFGGGTKVEIK(SEQ ID NO: 144) HC-86 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDIRYSPSLQGQVTISVDTSTSTAYLQWNSLKPSDTAMYYCARHGRGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 151) LC-86hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPITFGQGTKVEIK(SEQ ID NO: 152) HC-87 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 143)LC-87 hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPITFGQGTKVEIK(SEQ ID NO: 156) HC-88 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSLTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 158) LC-88hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPITFGQGTKVEIK(SEQ ID NO: 156) HC-89 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 160)LC-89 hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPITFGQGTKVEIK(SEQ ID NO: 152)Antibody HC-85/LC-85 (Ab 85)

The heavy chain variable region (VH) amino acid sequence of Ab85 isprovided below as SEQ ID NO: 143. The VH CDR amino acid sequences ofAb85 underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI(VH CDR3; SEQ ID NO: 147).

Ab85 VH sequence (SEQ ID NO: 143)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab85 isprovided below as SEQ ID NO 144. The VL CDR amino acid sequences of Ab85underlined below and are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO:148); DASNLET (VL CDR2; SEQ ID NO: 149); and QQANGFPLT (VL CDR3; SEQ IDNO: 150).

Ab85 VL sequence (SEQ ID NO: 144)DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGG GTKVEIKAntibody HC-86/LC-86 (Ab86)

The heavy chain variable region (VH) amino acid sequence of Ab86 isprovided below as SEQ ID NO: 151. The VH CDR amino acid sequences Ab86underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO: 145);IIYPGDSDIRYSPSLQG (VH CDR2; SEQ ID NO: 153); and HGRGYNGYEGAFDI (VHCDR3; SEQ ID NO: 3).

Ab86 VH sequence (SEQ ID NO: 151)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDIRYSPSLQGQVTISVDTSTSTAYLQWNSLKPSDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab86 isprovided below as SEQ ID NO 152. The VL CDR amino acid sequences of Ab86underlined below and are as follows: RASQGIGDSLA (VL CDR1; SEQ ID NO:154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3; SEQ IDNO: 155).

Ab86 VL sequence (SEQ ID NO: 152)DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-87/LC-87 (Ab87)

The heavy chain variable region (VH) amino acid sequence of Ab87 isprovided below as SEQ ID NO: 143. The VH CDR amino acid sequences ofAb87 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI(VH CDR3; SEQ ID NO: 147).

Ab87 VH sequence (SEQ ID NO: 143)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQGTLVTVSSThe light chain variable region (VL) amino acid sequence of Ab87 isprovided below as SEQ ID NO 156. The VL CDR amino acid sequences of Ab87underlined below and are as follows: RASQGIRNDLG (VL CDR1; SEQ ID NO:157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3; SEQ IDNO: 155).

Ab87 VL sequence (SEQ ID NO: 156)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-88/LC-88 (Ab88)

The heavy chain variable region (VH) amino acid sequence of Ab88 isprovided below as SEQ ID NO: 158. The VH CDR amino acid sequences ofAb88 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IIYPGDSLTRYSPSFQG (VH CDR2; SEQ ID NO: 159); and HGRGYNGYEGAFDI(VH CDR3; SEQ ID NO: 3).

Ab88 VH sequence (SEQ ID NO: 158)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSLTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab88 isprovided below as SEQ ID NO: 156. The VL CDR amino acid sequences ofAb88 underlined below and are as follows: RASQGIRNDLG (VL CDR1; SEQ IDNO: 157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3; SEQID NO: 155).

Ab88 VL sequence (SEQ ID NO: 156)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIKAntibody HC-89/LC-89 (Ab89)

The heavy chain variable region (VH) amino acid sequence of Ab89 isprovided below as SEQ ID NO: 160. The VH CDR amino acid sequences ofAb89 are underlined below and are as follows: NYWIG (VH CDR1; SEQ ID NO:145); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGRGYNGYEGAFDI (VHCDR3; SEQ ID NO: 3).

Ab89 VH sequence (SEQ ID NO: 160)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTLVTVSS

The light chain variable region (VL) amino acid sequence of Ab89 isprovided below as SEQ ID NO: 152. The VL CDR amino acid sequences ofAb89 underlined below and are as follows: RASQGIGDSLA (VL CDR1; SEQ IDNO: 154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3; SEQID NO: 155).

Ab89 VL sequence (SEQ ID NO: 152)DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQ GTKVEIK.

Example 11 In Vitro Binding Studies of Antibody 85, Antibody 86,Antibody 87, Antibody 88, and Antibody 89

Ab85, Ab86, Ab87, Ab88, and Ab89 were further studied for bindingcharacteristics using standard Octet binding. Antibody binding studieswere performed at 25 degrees celsius in 1× PBS supplemented with 0.1%w/v bovine serum albumin with a Pall ForteBio Octet Red96 using biolayerinterferometry (BLI). The indicated purified human antibody wasimmobilized onto anti-human Fc biosensors (AHC; Pall ForteBio 18-5063)and incubated with 33.3 nM (top traces) and 11 nM (bottom traces) CD117ectodomain (R&D Systems #332-SR). The resulting binding intervals, whichrepresented the association and dissociation curves were depicted inFIGS. 10A (i.e., HC-85/LC-85) and 10B (i.e., HC-1/LC-1). The apparentmonovalent affinity (K_(D)), apparent association rate (kon), andapparent dissociation rate (kdis) of the indicated purified IgG (i.e.,HC-85/LC-85) to purified human CD117 ectodomain (R&D Systems #332-SR)compared to a control (i.e., HC-1/LC-1) were depicted in Table 7. Theresults demonstrate a purified IgG (i.e., the HC-85/LC-85 IgG) bindswith high affinity to the purified human CD117 ectodomain and ischaracterized by a slow kdis (1/s) relative to the parent Ab1 antibody.Thus, despite the modification in the HCDR3, binding affinity wasimproved, i.e., with a slower dissociation rate, and the deamidationsite was removed.

TABLE 7 K_(D) (M) k_(on) (1/Ms) K_(dis)(1/s) HC-85/LC-85 (Ab85) 8.35 ×10⁻¹⁰ 5.20 × 10⁵ 4.34 × 10⁻⁴ HC-1/LC-1 (Ab1) 4.09 × 10⁻⁹  5.56 × 10⁵2.27 × 10⁻³

A comparison of the amino acid sequences of the heavy and light chainvariable regions and CDRs of Ab85 and Ab1 is described in FIGS. 11A and11B.

Example 12 Identification of Novel Anti-CD117 Antibodies

Having identified a number of improved anti-CD117 antibodies derivedfrom anti-CD117 antibody CK6, combinations of antigen binding regions(variable regions) from antibodies identified in Examples 8-11 were thentested for binding.

To identify further anti-CD117 antibodies with advantageous therapeuticproperties, heavy chain and light chain sequences disclosed here weremixed and matched to create novel anti-CD117 antibodies. The followingantibodies were identified from this process as preferred anti-CD117antibodies: HC-245/LC-245 (i.e., Ab245), HC-246/LC-246 (i.e., Ab246),HC-247/LC-247 (i.e., Ab247), HC-248/LC-248 (i.e., Ab248), andHC-249/LC-249 (i.e., Ab249).

TABLE 8 Antibody name Backbone Amino acid sequence HC-245 hIgG1EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 98)LC-245 hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQFNGYPLTFGQGTRLEIK(SEQ ID NO: 99) HC-246 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDI WGQGTMVTVSS (SEQ ID NO: 7) LC-246hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQFNGYPLTFGQGTRLEIK(SEQ ID NO: 99) HC-247 hIgG1 QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDI WGQGTMVTVSS (SEQ ID NO: 7) LC-247hKappa DIQMTQSPSSLSASVGDRVTITCRASRGISDYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQANSFPITFGQGTRLEIK(SEQ ID NO: 100) HC-248 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 98)LC-248 hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPLTFGQGTRLEIK(SEQ ID NO: 101) HC-249 hIgG1 EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 98)LC-249 hKappa DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQLNGYPLTFGQGTRLEIK(SEQ ID NO: 102)

FIGS. 11C and 11D provide an alignment of the heavy and light chainvariable regions of CK6 and Ab249.

Example 13 Kinetic Analysis of Affinity-Improved anti-CD117 Antibodies

To differentiate amongst the affinity matured anti-CD117 antibodies, themonovalent binding kinetics were determined by biolayer interferometryin comparison to the CK6 derivative Ab1 (HC-1, LC-1). HC-77/LC-77 (i.e.Ab77), HC-79/LC-79 (i.e. Ab79), HC-81/LC-81 (i.e. Ab81), HC-85/LC-85(i.e. Ab85), HC-245/LC-245 (i.e. Ab245), HC-246/LC-246 (i.e. Ab246),HC-247/LC-247 (i.e. Ab247), HC-248/LC-248 (i.e. Ab248), andHC-249/LC-249 (i.e. Ab249) and HC-1/LC-1 (i.e. Ab1) were immobilizedonto anti-human Fc biosensors (AHC; Pall ForteBio 18-5063) and incubatedwith 33 nM and 11 nM CD117 purified ectodomain (R&D Systems #332-SR).

The apparent monovalent affinity (KD), apparent association rate (Kon)and apparent dissociation rate (kdis) are tabulated in Table 9. For eachantibody the improvement in apparent dissociation rate compared toHC-1/LC-1 (i.e. Ab1) was calculated. The HC-249/LC-249 antibody (i.e.Ab249) demonstrated the most improved off-rate and the highest apparentmonovalent affinity. The HC-85/LC-85 antibody (i.e. Ab85) had thehighest apparent association rate amongst the set of antibodies testedin comparison to Ab1 and also had the deamidation site within the heavychain CDR3 removed. The resulting sensorgrams, which represent theassociation and dissociation curves, are shown for CK6 derivative(HC-1/LC-1, i.e. Ab1), the HC-85/LC-85 antibody (i.e. Ab85), and theHC-249/LC-249 antibody (i.e. Ab249) (FIGS. 12A, 12B and 12C).

TABLE 9 Fold improvement Antibody K_(D) (M) k_(on) (1/Ms) k_(dis) (1/s)(off-rate) Ab1 4.09 × 10⁹  5.56 × 10⁵ 2.27 × 10⁻³ — HC-77/LC-77 6.84 ×10⁻¹⁰ 3.15 × 10⁵ 2.15 × 10⁻⁴ 10.5 HC-79/LC-79 1.52 × 10⁻⁹  3.16 × 10⁵4.78 × 10⁻⁴ 4.7 HC-81/LC-81 8.77 × 10⁻¹⁰ 2.88 × 10⁵ 2.52 × 10⁻⁴ 9HC-85/LC-85 8.35 × 10⁻¹⁰ 5.20 × 10⁵ 4.34 × 10⁻⁴ 5.2 HC-245/LC-245 8.13 ×10⁻¹⁰ 3.50 × 10⁵ 2.85 × 10⁻⁴ 7.9 HC-246/LC-246 7.08 × 10⁻¹⁰ 3.61 × 10⁵2.55 × 10⁻⁴ 8.9 HC-247/LC-247 1.68 × 10⁻⁹  3.74 × 10⁵ 6.30 × 10⁻⁴ 3.6HC-248/LC-248 9.22 × 10⁻¹⁰ 3.46 × 10⁵ 3.19 × 10⁻⁴ 7.1 HC-249/LC-249 5.51× 10⁻¹⁰ 3.44 × 10⁵ 1.89 × 10⁻⁴ 12

As described in Table 9, the off rate of Ab249 was significantlyimproved over the parent antibody, Ab1, where the rate was about 12 foldslower than the parent.

Example 14 Characterization of Charge Variants of Anti-CD117 Antibodies

Capillary isoelectric focusing was performed on a subset ofaffinity-improved anti-CD117 antibodies to determine if sequencedifferentiation impacted the biophysical properties of the antibodies.Briefly, 10-40 micrograms of antibody were subjected to 7- and 15-daysincubation at 25 or 50 degrees Celsius and analyzed through a capillaryelectrophoresis method using the Maurice instrument manufactured byProtein Simple according to standard manufacturer instruction. Antibodysamples migrate to their electrically neutral pH. The fraction of acidicvariants was calculated based on absorbance peaks detected below theisoelectric point relative to the total injected sample.

The CK6 derivative Ab1 (HC-1/LC-1) exhibited 26% acidic species at thestart of the assay and this fraction increased to 57% and 54% of totalinjected antibody after 7 days of incubation at 25 and 50 degreesCelsius, respectively. With extended incubation for 15 days, thefraction of acidic variants for CK6 increased to 68% and 78% totalinjected antibody at 25 and 50 degrees Celsius, respectively.

In comparison, the HC-85/LC-85 antibody (i.e., Ab85) demonstratedsignificantly lower starting fractions of acidic variants (6.9% at TO)and reduced accumulation of acidic variants at 25 degrees Celsius (16%at day 7; 18% at day 15) and at 50 degrees Celsius (36% at day 7; 50% atday 15).

The HC-249/LC-249 antibody (i.e, Ab249) exhibited higher fractions ofacidic variants at the start of the experiment than Ab85 (31%), however,these fractions did not significantly increase following incubation at25 degrees Celsius (35% at day 7; 23% at day 15). After stress at 50degrees Celsius, the acidic species increased for Ab249 at both day 7and day 15 (52% and 65%; respectively).

The antibodies tested in this example were tested as IgG1 antibodieswith the same heavy and light constant regions described in SEQ ID NOs:169 and 183, respectively. Thus, the observed variation in stabilityreflected in the percentage of acidic variant as tested was due to thevariable regions.

Of all the antibodies analyzed, Ab85 had the lowest fraction of acidicvariants and the least accumulation of these species following stressconditions as described in FIGS. 13A and 13B.

Example 15 Characterization of Hydrophobicity of Anti-CD117 Antibodies

A subset of the affinity improved anti-CD117 antibodies was evaluatedafter incubation at 25 or 50 degrees Celsius for 15 days by hydrophobicinteraction chromatography (HIC). Briefly, 50 micrograms of theindicated antibody were injected onto a Tosoh TSKgel Phenyl-5PW 7.5 mmID×7.5 cm 10-micron column (Catalog #07573) on a Waters ARC HPLC/UPLCsystem. For the CK6 variant (Ab1; HC-1/LC-1), peak broadening wasobserved after 15 days of incubation at 25 and 50 degrees Celsius. Foraffinity improved antibodies HC-77/LC-77 (i.e., Ab 77), HC-79/LC-79(i.e., Ab79), and HC-81/LC-81 (i.e, Ab 81), significant peak broadeningwas evident in the chromatograms for both mild (25 degrees Celsius) andsevere (50 degrees Celsius) conditions. Ab85 demonstrated minimal peakbroadening after incubation at 25 or 50 degrees Celsius after 15 days,exhibiting the lowest change in hydrophobicity of the affinity improvedanti-CD117 antibodies tested and compared to the CK6 variant Ab1(HC-1/LC-1) as described in FIGS. 14A-14E. As described in Example 14,the antibodies contained the same constant region sequences.

Example 16 In Vitro Analysis of Affinity Improved Anti-CD117-ADCs Usingan In Vitro Cell Killing Assay

To evaluate the potency of affinity improved anti-CD117 antibodies asADCs, Kasumi-1 cells were cultured for three days in the presence of theindicated anti-CD117-ADC (antibody conjugated to an amatoxin) or thecontrol isotype ADC. Cell viability was measured by Celltiter Glo (FIG.15).

To examine the potency of affinity improved anti-CD117 antibodies asADCs on primary cells in vitro, human HSCs (i.e., isolated primary humanCD34+ selected Bone Marrow Cells (BMCs), human CD34+ BMCs were culturedfor five days with the indicated anti-CD117-ADC or the control isotypeADC in the presence of IL-6, TPO, and FLT-3 ligand. CD34+ CD90+ cellcounts were determined by flow cytometry (FIG. 16).

As shown in FIGS. 15 and 16, both the HC-85/LC-85 ADC (i.e, Ab85) andthe HC-249/LC-249 ADC (i.e, Ab249) were effective at killing Kasumi-1(1.76×10⁻¹¹ M and 2.5×10⁻¹¹M, respectively; see Table 10) and primaryhuman CD34+ cells in vitro (5.74×10⁻¹² M and 6.67×10⁻¹²M, respectively;see Table 11) and the potency was similar to CK6 (1.48×10⁻¹¹ M inKasumi-1; 9.8×10⁻¹²M in primary human CD34+ CD90+ cells) (Tables 10 and11).

Thus, the anti-CD117 antibodies Ab85 and Ab249 exhibited markedimprovements in monovalent affinity (FIGS. 12B and 12C), superiorbiophysical behavior as measured by acidic variants and hydrophobicityunder thermal stress (FIGS. 13A and 13B and FIGS. 14A-14E), and similarin vitro potency as ADCs in cytoxicity assays on Kasumi-1 (FIG. 15) andprimary human CD34+ CD90+ cells when compared to CK6 (FIG. 16). Further,Ab85 had an improved HC CDR3 domain as a potential deamidation site wasremoved without negatively impacting the characteristics of theantibody.

TABLE 10 Antibody IC50 (M) CK6 1.48 × 10⁻¹¹ HC-249/LC-249 2.50 × 10⁻¹¹HC-85/LC-85 1.76 × 10⁻¹¹

TABLE 11 Antibody IC50 % efficacy CK6 9.77 × 10⁻¹² 70.80 HC-77/LC-776.94 × 10⁻¹² 54.38 HC-79/LC-79 7.09 × 10⁻¹² 63.37 HC-81/LC-81 3.77 ×10⁻¹² 68.74 HC-85/LC-85 5.74 × 10⁻¹² 71.18 HC-245/LC-245 5.78 × 10⁻¹²76.43 HC-246/LC-246 4.53 × 10⁻¹² 67.82 HC-247/LC-247 8.37 × 10⁻¹² 55.43HC-248/LC-248 6.46 × 10⁻¹² 47.80 HC-249/LC-249 6.67 × 10⁻¹² 52.01

Example 17 In Vivo HSC Depletion Assay Using an Affinity ImprovedAnti-CD117 ADCs

An in vivo experiment was performed to evaluate the affinity improvedantibody HC-85/LC-85 (i.e, Ab85) with an engineered Fc (i.e., an H435AFc mutation). The H435A mutation is associated with a decrease in halflife in antibodies in which it is introduced. This experiment wasperformed to determine how this mutation would impact the overallcharacteristics of an Ab85 ADC (Ab85 conjugated to an amatoxin via acleavable linker).

In vivo HSC depletion assays were conducted using humanized NSG mice(purchased from Jackson Laboratories). The Fc-modified Ab85 antibody(i.e., characterized by a H435A Fc mutation) was conjugated as an ADCand administered as a single injection of 0.3 mg/kg to the humanizedmouse model. Blood was collected on days 0, 7, and 14 and examined byflow cytometry. The percentage of human CD33+ of treated or controltreated mice relative to baseline are shown (FIG. 17A). Bone marrow wascollected on day 14 and the absolute number of CD34+ cells wasdetermined by flow cytometry (FIG. 17B).

The results indicate that humanized NSG mice treated with theFc-modified Ab85 ADC showed significant depletion of human CD33+ myeloidcells relative to baseline, 7 and 14 days following a singleadministration of the treatment regimen and indicate that theFc-modified Ab85 ADC depleted myeloid cells as a result of the depletionof early progenitor cells. In addition, humanized NSG mice treated withthe Ab85 ADC showed significant depletion of human HSCs in the bonemarrow, 14 days following a single administration of the ADC whencompared to the control. Thus, Ab85 ADC with a modified Fc region waseffective at selectively depleting CD34+ stem cells and CD33+ progenitorcells.

Example 18 CD117-Amanitin Antibody Drug Conjugates Effectively DepleteHuman and Non-Human Primate HSCs

Genotoxic conditioning prior to allogeneic and autologous hematopoieticstem cell (HSC) transplantation (HSCT) and the infusion of genecorrected autologous HSCs limits the use of these potentially curativetreatments due to risks of regimen-related morbidities and mortality,including risks of infertility and secondary malignancies. CD117, whichis specifically expressed on HSCs and progenitors is rapidlyinternalized and is an ideal target for an ADC-based approach toconditioning. As it has been previously shown that a single dose of ananti-CD117 ADC depleted >95% of bone marrow HSCs in a humanized mousemodel and reduced disease burden while extending survival in an AMLtumor model (Hartigan et al., Blood 2017 130:1894), the aim of thisexample was to develop a potent anti-CD117 ADC highly effective ineliminating host HSC with a short half-life and minimal adverse sideeffects in a non-human primate (NHP) model.

The cytotoxic payloads a calicheamicin variant, a pyrrolobenzodiazepine(PBD), and an amanitin (AM) were site specifically conjugated to ananti-CD117 antibody (CK6; wild type half-life). The ADCs were titratedand evaluated for in vitro cytotoxicity using human bone marrow CD34+cells (FIG. 18). The ADCs were administered in ascending doses tohumanized NSG mice. HSC depletion and immunophenotype of the human cellsin the peripheral blood was determined by flow cytometry at day 21 (datanot shown). Anti-CD117 conjugated with the RNA polymerase II inhibitoramanitin (AM) resulted in >90% depletion of human HSCs in humanized NSGmice at 0.3 mg/kg. The AM-conjugates also demonstrated a broadtherapeutic window in this model (therapeutic index of >120). These dataalso show that the calicheamicin and amanitin ADCs demonstratecomparable depletion of HSCs.

Amanitin-conjugate mediated NHP HSC depletion was evaluated in malecynomolgus monkeys in single ascending doses (3/group). HSC content inthe bone marrow was monitored by flow cytometry and colony-forming unit(CFU) analysis on day 7 or 14 and 56 post dosing. Hematology andclinical chemistries were evaluated throughout the two-month study.

On-target, dose dependent decreases in phenotypic HSCs and CFUs wereobserved in the bone marrow at day 7 post anti-CD117-AM dosing with >95%HSC depletion observed with a single dose of 0.3 mg/kg (FIG. 19). In theperiphery, a dose dependent transient decrease in reticulocytes wasobserved at day 4 with a neutrophil and monocyte nadir at day 18 (datanot shown). The severity and duration of the depletion was also dosedependent. The anti-CD117-AM induced depletion was on target andamanitin dependent as the unconjugated antibody and isotype-AM had noeffect (FIG. 19). Notably, white blood cell and lymphocyte counts werestable through day 56 (data not shown), demonstrating that this strategywill spare the adaptive immune system. Platelet nadir occurred 4-8 dayspost infusion and was dose dependent transient and reversible. This alsooccurred with the isotype-AM suggesting the effect was off-target.

Because the half-life of the anti-CD117-AM was 5 days, a second doseescalation study with anti-CD117-AM (HC-85; LC-85) engineered to have ashort half-life (˜18 h) was performed in NHP, as this may be moresuitable for the transplant setting. The short half-life anti-CD117-AMdemonstrated similar potency on all cell parameters at 0.3 mg/kg or witha fractionated dose (0.3/0.2 mg/kg Q3Dx2) and was well tolerated at theeffective dose (FIG. 20). As expected, the fast half-life anti-CD117-AMwas rapidly cleared with a half-life of 15-18 h. In conclusion,anti-CD117-AM exhibits potent elimination of NHP HSCs and progenitors invivo.

Example 19 Anti-CD117 Antibody Drug Conjugates (ADCs) ConditioningAgents Have Profound In Vivo Anti-Leukemia Activity in Cell Line andPatient Derived Xenograft Models

Allogeneic hematopoietic stem cell transplant (HSCT) is a potentiallycurative approach in patients with refractory or high risk hematologicmalignancies. Prior to transplant, patients are conditioned with highdose chemotherapy or chemotherapy and total body irradiation which areassociated with early and late morbidities and substantial risk ofmortality. As a result, many eligible patients do not considertransplant and of those transplanted, ⅔ can only tolerate a reducedintensity conditioning which is associated with increased relapse rates.Thus, safer and more effective conditioning agents with improved diseasecontrol are urgently needed. To meet this need, we developed a novelantibody drug conjugate (ADC) conjugated to amanitin (AM) targetingCD117 which is expressed on hematopoietic stem and progenitor cells andacute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cellsin >60% of patients. The aim of this example was to determine theanti-leukemia potency of anti-CD117 conjugated to amanitin, an agentpreviously shown to deplete primary human HSPCs in vitro and in vivo(Hartigan et al. Blood. 130; 1894 (2017)).

An ADC comprising the anti-CD117 antibody CK6 was conjugated toamanitin. The ADC was tested in three patient-derived xenografts (PDX)developed from FLT-3+NPM1+ AML samples (J000106132 (FIG. 21A),J000106565 (FIG. 21B), J000106134 (FIG. 21C)) with varying growthkinetics (median survival of vehicle treated groups was 43 days (Table12; see also FIG. 21A), 63 days (Table 13; see also FIG. 21B), 82 days(Table 14; see also FIG. 21C) post inoculation) that express CD117(Jackson Laboratories).

TABLE 12 ADC Dose (mg/kg) Median survival (Days) PBS — 43 Anti-CD117-AM1 75.5 Anti-CD117-AM 0.3 55 Isotype-AM 1 46 Anti-CD117-naked 1 43

TABLE 13 ADC Dose (mg/kg) Median survival (Days) PBS — 82 Anti-CD117-AM1 >225 Anti-CD117-AM 0.3 179 Isotype-AM 1 75 Anti-CD117-naked 1 75

TABLE 14 ADC Dose (mg/kg) Median survival (Days) PBS — 63 Anti-CD117-AM1 106 Anti-CD117-AM 0.3 95 Isotype-AM 1 63 Anti-CD117-naked 1 63

For the three PDX (J000106132 (FIG. 21A), J000106565 (FIG. 21B),J000106134 (FIG. 21C)), a single intravenous dose of ADCs (CD117-AM,isotype-AM (ISO-AM), unconjugated anti-CD117 antibody, or vehicle PBS)were administered to AML-PDX animals when 2-5% blasts were observed inthe blood. With 4-5 mice/group/AML-PDX model, survival was significantlyincreased in recipients of 0.3 mg/kg CD117-AM and 1 mg/kg CD117-AM ascompared to vehicle controls (Tables 12-14; FIGS. 21A-21C).

In addition to depletion of normal host HSPC, CD117-AM is a potentanti-leukemia agent based on these data in humanized murine models withestablished AML. Together with prior reports on the potency of CD117-AMas a conditioning agent, this non-genotoxic ADC (or variants of CK6 withimproved properties described herein) may be useful in patients withactive disease and in recipients of reduced dose conditioning who are athigh risk of disease relapse.

Example 20 Identification of Novel Anti-CD117 Antibodies by RatImmunization

As a third strategy, to identify novel anti-CD117 antibodies, 5 ratswere immunized with synthetic DNA coding for human CD117 ectodomain(aa26-524; P010721) and hybridoma fusions were prepared according tostandard methods known in the art. Hybridomas were screened for bindingto cell lines expressing human CD117 ectodomain (aa26-524; P010721) andcynomologus monkey CD117 ectodomain (aa26-521; F6V858) to identifyantibodies which would proceed in screening. The following antibodieswere identified as positive binders to both cell lines by flowcytometery: HC-17/LC-17 (i.e., Ab17), HC-18/LC-18 (i.e., Ab18),HC-19/LC-19 (i.e., Ab19), HC-20/LC-20 (i.e., Ab20), HC-21/LC-21 (i.e.,Ab21), HC-22/LC-22 (i.e., Ab22), HC-23/LC-23 (i.e., Ab23), HC-24/LC-24(i.e., Ab24), HC-25/LC-25 (i.e., Ab25), HC-27/LC-27 (i.e., Ab27), andHC-28/LC-28 (i.e., Ab28).

To confirm binding to the desired target, purified antibodies wereanalyzed for binding to purified recombinant CD117 ectodomain bybio-layer interferometry. Binding analysis of the antibodies identifiedfrom the rat immunizations reveals a wide set of association anddissociation kinetics as demonstrated in FIGS. 22 and 23. The apparentkinetic values are provided in Table 4 and Table 15. Table 15 provides atable listing the apparent monovalent affinity (K_(D)), apparentassociation rate (k_(on)), and apparent dissociation rate (k_(dis)) ofthe indicated purified IgG to purified rhesus CD117 ectodomain. Asprovided above, Table 4 provides a table listing the apparent monovalentaffinity (K_(D)), apparent association rate (k_(on)), and apparentdissociation rate (k_(dis)) the indicated purified IgG (including theantibodies identified in this Example) to purified human CD117ectodomain.

TABLE 15 K_(D) (M) k_(on) (1/Ms) k_(dis) (1/s) 017 (Ab17) 5.62E−103.07E+05 1.73E−04 018 (Ab18) 5.38E−08 2.21E+05 1.19E−02 019 (Ab19)2.34E−09 1.77E+05 4.15E−04 020 (Ab20) 1.39E−09 4.21E+05 5.85E−04 021(Ab21) 8.69E−10 4.10E+05 3.57E−04 022 (Ab22) 8.91E−11 1.74E+06 1.55E−04023 (Ab23) 7.62E−09 2.80E+05 2.14E−03 024 (Ab24) 1.96E−08 3.97E+047.77E−04 025 (Ab25) 8.10E−10 2.41E+06 1.95E−03 027 (Ab27) 8.90E−103.09E+05 2.75E−04 028 (Ab28) 3.60E−09 5.21E+05 1.87E−03

To confirm the species cross-reactivity of the antibodies identified inthe screening of the rat hybridomas, purified antibodies were analyzedfor binding to purified rhesus CD117 ectodomain by bio-layerinterferometry. Binding analysis of the antibodies demonstrates strongcross-reactivity for the set of antibodies. Analysis of the apparentkinetic values for binding to rhesus antigen (Table 15) demonstratesstrong correlation with the apparent association and dissociation ratesobserved for binding to the human antigen (Table 4).

TABLE 16 AMINO ACID SEQUENCE SUMMARY Sequence Identifier DescriptionAmino Acid Sequence SEQ ID NO: 1 CK6 CDR-H1 SYWIG SEQ ID NO: 2 CK6CDR-H2 IIYPGDSDTRYSPSFQG Ab249 CDR-H2 SEQ ID NO: 3 CK6 CDR-H3HGRGYNGYEGAFDI SEQ ID NO: 4 CK6 CDR-L1 RASQGISSALA SEQ ID NO: 5 CK6CDR-L2 DASSLES SEQ ID NO: 6 CK6 CDR-L3 CQQFNSYPLT SEQ ID NO: 7 Heavychain QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-1SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID NO: 8Light chain variable AIQLTQSPSSLSASVGDRVTITCRASQGVSSALAWY region of LC-1QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKSEQ ID NO: 7 Heavy chain QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variableregion of WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-2SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID NO: 9Light chain variable DIQLTQSPSSLSASVGDRVTITCRASQGIRTDLGWY region of LC-2QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKSEQ ID NO: 7 Heavy chain QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variableregion of WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-3SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIRMTQSPSSLSASVGDRVTITCRASQGIRNDLAWY NO: 10 region ofLC-3 QQKPGKTPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-4SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 11 region ofLC-4 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVDIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-5SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable NIQMTQSPSSLSASVGDRVTITCRASQAISDYLAWF NO: 12 region ofLC-5 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-6SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIRMTQSPSSLSASVGDRVIIACRASQGIGGALAWY NO: 13 region ofLC-6 QQKPGNAPKVLVYDASTLESGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-7SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIAMTQSPPSLSAFVGDRVTITCRASQGIISSLAWYQ NO: 14 region ofLC-7 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTIRSLQPEDFATYYCQQFNSYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-8SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWY NO: 15 region ofLC-8 QQKAGKAPKVLISDASSLESGVPSRFSGSGSGTDFTLSISSLQPEDFATYYCQQFNGYPLTFGGGTKVDIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-9 amino acidSAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG sequence YEGAFDIWGQGTMVTVSS SEQ IDLight chain variable AIRMTQSPSSLSASVGDRVTITCQASQGIRNDLGWY NO: 16 regionof LC-9 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFNSYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-10SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable NIQMTQSPSSLSTSVGDRVTITCRASQGIGTSLAWY NO: 17 region ofLC-10 QQKPGKPPKLLIYDASSLESGVPSRLSGSGSGTDFTLTISSLQPEDFATYYCQQSNSYPITFGQGTRLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-11SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIQLTQSPSSLSASVGDRVTITCRASQSIGDYLTWYQ NO: 18 region ofLC-11 QKPGKAPKVLIYGASSLQSGVPPRFSGSGSGTDFTLTVSSLQPEDFATYYCQQLNSYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-12SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVRSTLAWY NO: 19 region ofLC-12 QQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNGYPLTFGQGTRLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-13SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 20 region ofLC-13 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-14SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGISSFLAWYQ NO: 21 region ofLC-14 QKPGKAPKLLIYDASTLQSGVPSRFSGSASGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-15SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIQLTQSPSSLSASVGDRVTITCRASQGIGSALAWYQ NO: 22 region ofLC-15 QKPGIGPKLLIYDASTLESGVPARFSGSGSRTDFTLTITSLQPEDFATYYCQQFNGYPLTFGGGTKLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-16SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable AIQLTQSPSSLSASVGDRVTITCRASQGITSALAWYQ NO: 23 region ofLC-16 EKPGKAPNLLIYDASSLESGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVDIK SEQ ID Heavy chainQIQLVQSGPELRKPGESVKISCKASGYTFTDYAMYW NO: 24 variable region ofVKQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFS HC-17LEASANTANLQISNLKNEDTATYFCARARGLVDDYV MDAWGQGTSVTVSS SEQ ID Light chainvariable SYELIQPPSASVTLGNIVSLICVGDELSKRYAQWYQ NO: 25 region of LC-17QKPDKTIVSVIYKDSERPSGISDRFSGSSSGTTATLTIHGTLAEDEADYYCLSTYSDDNLPVFGGGTKLTVL SEQ ID Heavy chainEVQLQQYGAELGKPGTSVRLSCKVSGYNIRNTYIHW NO: 26 variable region ofVNQRPGEGLEWIGRIDPTNGNTISAEKFKTKATLTAD HC-18TSSHTAYLQFSQLKSDDTAIYFCALNYEGYADYWGQ GVMVTGSS SEQ ID Light chainvariable DIQMTQSPSFLSASVGDRVTINCKASQNINKYLNWY NO: 27 region of LC-18QQKVGEAPKRLIFKTNSLQTGIPSRFSGSGSGTDYT LTISSLQTEDVATYFCFQYNIGYTFGAGTKVELKSEQ ID Heavy chain EVQLQESGPGLVKPSQSLSLTCSVTGYSISSNYRWN NO: 28 variableregion of WIRKFPGNKVEWMGYINSAGSTNYNPSLKSRISMTR HC-19DTSKNQFFLQVNSVTTEDTATYYCARSLRGYITDYS GFFDYWGQGVMVTVSS SEQ ID Light chainvariable DIRMTQSPASLSASLGETVNIECLASEDIFSDLAWYQ NO: 29 region of LC-19QKPGKSPQLLIYNANSLQNGVPSRFSGSGSGTRYSL KINSLQSEDVATYFCQQYKNYPLTFGSGTKLEIKSEQ ID Heavy chain EVQLQQYGAELGKPGTSVRLSCKLSGYKIRNTYIHW NO: 30 variableregion of VNQRPGKGLEWIGRIDPANGNTIYAEKFKSKVTLTAD HC-20TSSNTAYMQLSQLKSDDTALYFCAMNYEGYEDYWG QGVMVTVSS SEQ ID Light chainvariable DIQMTQSPSFLSASVGDSVTINCKASQNINKYLNWY NO: 31 region of LC-20QQKLGEAPKRLIHKTDSLQTGIPSRFSGSGSGTDYT LTISSLQPEDVATYFCFQYKSGFMFGAGTKLELKSEQ ID Heavy chain QIQLVQSGPELKKPGESVKISCKASGYTFTDYAVYW NO: 32 variableregion of VIQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSL HC-21ETSASTANLQISNLKNEDTATYFCARGAGMTKDYVM DAWGRGVLVTVS SEQ ID Light chainvariable SYELIQPPSASVTLGNTVSLTCVGDELSKRYAQWYQ NO: 33 region of LC-21QKPDKTIVSVIYKDSERPSDISDRFSGSSSGTTATLTIHGTLAEDEADYYCLSTYSDDNLPVFGGGTKLTVL SEQ ID Heavy chainQVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYLVHW NO: 34 variable region ofVRQPPGKTLEWVGLMWNDGDTSYNSALKSRLSISR HC-22DTSKSQVFLKMHSLQAEDTATYYCARESNLGFTYW GHGTLVTVSS SEQ ID Light chainvariable DIQMTQSPASLSASLEEIVTITCKASQGIDDDLSWYQ NO: 35 region of LC-22QKPGKSPQLLIYDVTRLADGVPSRFSGSRSGTQYSL KISRPQVADSGIYYCLQSYSTPYTFGAGTKLELKSEQ ID Heavy chain EVQLQQYGAELGKPGTSVRLSCKVSGYNIRNTYIHW NO: 36 variableregion of VHQRPGEGLEWIGRIDPTNGNTISAEKFKSKATLTAD HC-23TSSNTAYMQFSQLKSDDTAIYFCAMNYEGYADYWG QGVMVTVSS SEQ ID Light chainvariable DIQMTQSPSFLSASVGDRLTINCKASQNINKYLNWY NO: 37 region of LC-23QQKLGEAPKRLIFKTNSLQTGIPSRFSGSGSGTDYTL TISSLQPEDVATYFCFQYNIGFTFGAGTKLELKSEQ ID Heavy chain EVQLVESGGGLVQSGRSLKLSCAASGFTVSDYYMA NO: 38 variableregion of WVRQAPTKGLEWVATINYDGSTTYHRDSVKGRFTIS HC-24RDNAKSTLYLQMDSLRSEDTATYYCARHGDYGYHY GAYYFDYWGQGVMVTVSS SEQ ID Lightchain variable DIVLTQSPALAVSLGQRATISCRASQTVSLSGYNLIH NO: 39 region ofLC-24 WYQQRTGQQPKLLIYRASNLAPGIPARFSGSGSGTDFTLTISPVQSDDIATYYCQQSRESWTFGGGTNLEMK SEQ ID Heavy chainQIQLVQSGPELKKPGESVKISCKASGYTFTDYAIHWV NO: 40 variable region ofKQAPGQGLRWMAWINTETGKPTYADDFKGRFVFSL HC-25EASASTAHLQISNLKNEDTATFFCAGGSHWFAYWG QGTLVTVSS SEQ ID Light chainvariable SYELIQPPSASVTLENTVSITCSGDELSNKYAHWYQ NO: 41 region of LC-25QKPDKTILEVIYNDSERPSGISDRFSGSSSGTTAILTIRDAQAEDEADYYCLSTFSDDDLPIFGGGTKLTVL SEQ ID Heavy chainQIQLVQSGPELKKPGESVKISCKASGYTFTDYAVYW NO: 32 variable region ofVIQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSL HC-26ETSASTANLQISNLKNEDTATYFCARGAGMTKDYVM DAWGRGVLVTVS SEQ ID Light chainvariable SYELIQPPSTSVTLGNTVSLTCVGNELPKRYAYWFQ NO: 42 region of LC-26QKPDQSIVRLIYDDDRRPSGISDRFSGSSSGTTATLTIRDAQAEDEAYYYCHSTYTDDKVPIFGGGTKLTVL SEQ ID Heavy chainEVQLVESGGGLVQPGRSMKLSCKASGFTFSNYDMA NO: 43 variable region ofWVRQAPTRGLEWVASISYDGITAYYRDSVKGRFTIS HC-27RENAKSTLYLQLVSLRSEDTATYYCTTEGGYVYSGP HYFDYWGQGVMVTVSS SEQ ID Light chainvariable DIQMTQSPSSMSVSLGDTVTITCRASQDVGIFVNWF NO: 44 region of LC-27QQKPGRSPRRMIYRATNLADGVPSRFSGSRSGSDY SLTISSLESEDVADYHCLQYDEFPRTFGGGTKLELKSEQ ID Heavy chain EVQLQQYGAELGKPGTSVRLSCKVSGYKIRNTYIHW NO: 45 variableregion of VNQRPGKGLEWIGRIDPANGNTIYAEKFKSKVTLTAD HC-28TSSNTAYMQLSQLKSDDTALYFCAMNYEGYEDYWG QGVMVTVSS SEQ ID Light chainvariable DIQMTQSPSFLSASVGDSVTINCKASQNINKYLNWY NO: 46 region of LC-28QQKLGEAPKRLIHKTNSLQPGFPSRFSGSGSGTDYT LTISSLQPEDVAAYFCFQYNSGFTFGAGTKLELKSEQ ID Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIH NO: 47 variableregion of WVRQAPGQGLEWMGWMNPHSGDTGYAQKFQGR HC-29VTMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRG YNGYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIGNELGWY NO: 48 region ofLC-29 QQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPLTFGQGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLH NO: 49 variable region ofWVRQAPGQGLEWMGWINPNSGDTNYAQNFQGRV HC-30TMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGY NGYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 50 region ofLC-30 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGGGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLH NO: 51 variable region ofWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRV HC-31TMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGY EGYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 52 region ofLC-31 QQKPGKAPKLLIYDASELETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIH NO: 53 variable region ofWVRQAPGQGLEWMGWLNPSGGGTSYAQKFQGRV HC-32TMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGY DGYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 54 region ofLC-32 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGGGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFSTYYMH NO: 55 variable region ofWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVT HC-33MTRDTSTSTVYMKLSSLRSEDTAVYYCARHGRGYE GYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWY NO: 56 region ofLC-33 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIH NO: 57 variable region ofWVRQAPGQGLEWMGIINPSGGNTNYAQNFQGRVT HC-34MTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYN AYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 58 region ofLC-34 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVNGYPLTFGGGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAIS NO: 59 variable region ofWVRQAPGQGLEWMGVINPTVGGANYAQKFQGRVT HC-35MTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYN EYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCQASQDISDYLNWY NO: 60 region ofLC-35 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPLTFGGGTKLEIK SEQ ID Heavy chainQVQLVQSGAEVKKLGASVKVSCKASGYTFSSYYMH NO: 61 variable region ofWVRQAPGQGLEWMGVINPNGAGTNFAQKFQGRVT HC-36MTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYE GYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 50 region ofLC-36 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGGGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMH NO: 62 variable region ofWVRQAPGQGLEWMGWINPTGGGTNYAQNFQGRV HC-37TMTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGY EGYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDVSWY NO: 63 region ofLC-37 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLSGYPITFGQGTKLEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIH NO: 64 variable region ofWVRQAPGQGLEWMGMINPSGGSTNYAQKFQGRVT HC-38MTRDTSTSTVYMELSSLRSEDTAVYYCARHGRGYN DYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQSISDWLAWY NO: 65 region ofLC-38 QQKPGKAPKLLIYEASNLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKVEIK SEQ ID Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYIFSAYYIHW NO: 66 variable region ofVRQAPGQGLEWMGIINPSGGSTRYAQKFQGRVTMT HC-39RDTSTSTVYMELSSLRSEDTAVYYCARHGRGYGGY EGAFDIWDQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIGDYVAWY NO: 67 region ofLC-39 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQGTRLEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYRFTSYWIG NO: 68 variable region ofWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTI HC-40SVDKSNSTAYLQWSSLKASDTAMYYCARHGRGYN GYEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWY NO: 69 region ofLC-40 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTYFTLTISSLQPEDFATYYCQQGASFPITFGQGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGSSFPNSWIAW NO: 70 variable region ofVRQMPGKGLEWMGIIYPSDSDTRYSPSFQGQVTISA HC-41DKSISTAYLQWSSLEASDTAMYYCARHGRGYNGYE GAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWY NO: 71 region ofLC-41 QQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIG NO: 72 variable region ofWVRQMPGKGLEWMGIMYPGDSDTRYSPSFQGQVT HC-42ISADKSISTAYLQWSSLKASDTAMYYCARHGRGYNA YEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQSINNWLAWY NO: 73 region ofLC-42 QQKPGKAPKLLIYDAFILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQLNSYPLTFGPGTKVDIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFTNWIAWV NO: 74 variable region ofRQMPGKGLEWMGIIYPGDSETRYSPSFQGQVTISAD HC-43KSISTAYLQWSSLKASDTAMYYCARHGRGYYGYEG AFDIWGQGTLVTVSSAS SEQ ID Light chainvariable DIQMTQSPSSLSASVGDRVTITCRASQGISDNLNWY NO: 75 region of LC-43QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQAISFPLTFGQGTKVEIKSEQ ID Heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYNFTSYWIG NO: 76 variableregion of WVRQMPGKGLEWMGVIYPDDSETRYSPSFQGQVTI HC-44SADKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASRDIRDDLGWY NO: 77 region ofLC-44 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYTFNTYIGWV NO: 78 variable region ofRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQVTISA HC-45DKAISTAYLQWSSLKASDTAMYYCARHSRGYNGYE GAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWY NO: 79 region ofLC-45 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPVTFGQGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYNFTTYWIGW NO: 80 variable region ofVRQMPGKGLEWMGIIHPADSDTRYNPSFQGQVTISA HC-46DKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYE GAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRVSQGISSYLAWY NO: 81 region ofLC-46 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYRFSNYWIA NO: 82 variable region ofWVRQMPGKGLEWMGIIYPDNSDTRYSPSFQGQVTI HC-47SADKSISTAYLQWSSLKASDTAMYYCARHGRGYDG YEGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRSDLAWY NO: 83 region ofLC-47 QQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLSFGQGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYRFASYWIG NO: 84 variable region ofWVRQMPGKGLEWMGITYPGDSETRYNPSQGQVTIS HC-48ADKSISTAYLQWSSLKASDTAMYYCARHGRGYGGY EGAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 85 region ofLC-48 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW NO: 86 variable region ofVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA HC-49DKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYE GAFDIWGQGTLVTVSSAS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWY NO: 87 region ofLC-49 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNSFPLTFGQGTRLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-74SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQ NO: 88 region ofLC-74 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-75SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQ NO: 89 region ofLC-75 QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-76SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWY NO: 90 region ofLC-76 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-77SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQ NO: 91 region ofLC-77 QKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-78SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQ NO: 92 region ofLC-78 QKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-79SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWY NO: 93 region ofLC-79 QQKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-80SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ NO: 94 region ofLC-80 QKPGKAPKLLIYDASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-81SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVISALAWYQ NO: 95 region ofLC-81 QKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-82SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQ NO: 96 region ofLC-82 QKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-83SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWY NO: 97 region ofLC-83 QQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-84SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQLTQSPSSLSASVGDRVTITCRASQGVGSALAWY NO: 97 region ofLC-84 QQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 98 variable region ofVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA HC-245DKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE GAFDIWGQGTLVTVSS SEQ ID Light chainvariable DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWY NO: 99 region of LC-245QQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNGYPLTFGQGTRLEIKSEQ ID NO: 7 Heavy chain QVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variableregion of WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-246SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWY NO: 99 region ofLC-246 QQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNGYPLTFGQGTRLEIK SEQ ID NO: 7 Heavy chainQVQLVQSGAAVKKPGESLKISCKGSGYRFTTYWIG variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI HC-247SAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTMVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASRGISDYLAWY NO: 100 region ofLC-247 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 98 variable region ofVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA HC-248DKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE GAFDIWGQGTLVTVSS SEQ ID Light chainvariable DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWY NO: 101 region of LC-248QQKPGKAPKLLIYDASTLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIKSEQ ID Heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 98 variableregion of VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA HC-249DKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE GAFDIWGQGTLVTVSS SEQ ID Light chainvariable DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWY NO: 102 region of LC-249QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIKSEQ ID Heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 143 variableregion of WVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI Ab 85SADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG YEGAFDIWGQGTLVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWY NO: 144 region of Ab85 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK SEQ ID Ab85 CDR-H1 NYWIG NO: 145 SEQID Ab85 CDR-H2 IINPRDSDTRYRPSFQG NO: 146 SEQ ID Ab85 CDR-H3HGRGYEGYEGAFDI NO: 147 SEQ ID Ab85 CDR-L1 RSSQGIRSDLG NO: 148 SEQ IDAb85 CDR-L2 DASNLET NO: 149 Ab249 CDR-L2 SEQ ID Ab85 CDR-L3 QQANGFPLTNO: 150 SEQ ID Heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 151variable region of WVRQMPGKGLEWMGIIYPGDSDIRYSPSLQGQVTIS Ab 86VDTSTSTAYLQWNSLKPSDTAMYYCARHGRGYNGY EGAFDIWGQGTLVTVSS SEQ ID Light chainvariable DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWY NO: 152 region of Ab 86QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQLNGYPITFGQGTKVEIKSEQ ID Ab86 CDR-H1 NYWIG NO: 145 SEQ ID Ab86 CDR-H2 IIYPGDSDIRYSPSLQGNO: 153 SEQ ID NO: 3 Ab86 CDR-H3 HGRGYNGYEGAFDI SEQ ID Ab86 CDR-L1RASQGIGDSLA NO: 154 SEQ ID Ab86 CDR-L2 DASNLET NO: 149 SEQ ID Ab86CDR-L3 QQLNGYPIT NO: 155 SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 143 variable region ofWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI Ab 87SADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG YEGAFDIWGQGTLVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 156 region of Ab87 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQGTKVEIK SEQ ID Ab87 CDR-H1 NYWIG NO: 145 SEQID Ab87 CDR-H2 IINPRDSDTRYRPSFQG NO: 146 SEQ ID Ab87 CDR-H3HGRGYEGYEGAFDI NO: 147 SEQ ID Ab87 CDR-L1 RASQGIRNDLG NO: 157 SEQ ID NO:5 Ab87 CDR-L2 DASSLES SEQ ID Ab87 CDR-L3 QQLNGYPIT NO: 155 SEQ ID Heavychain EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 158 variable region ofWVRQMPGKGLEWMGIIYPGDSLTRYSPSFQGQVTI Ab 88SADKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTLVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY NO: 156 region of Ab88 QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQGTKVEIK SEQ ID Ab88 CDR-H1 NYWIG NO: 145 SEQID Ab88 CDR-H2 IIYPGDSLTRYSPSFQG NO: 159 SEQ ID NO: 3 Ab88 CDR-H3HGRGYNGYEGAFDI SEQ ID Ab88 CDR-L1 RASQGIRNDLG NO: 157 SEQ ID NO: 5 Ab88CDR-L2 DASSLES SEQ ID Ab88 CDR-L3 QQLNGYPIT NO: 155 SEQ ID Heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 160 variable region ofWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI Ab89SADKSISTAYLQWSSLKASDTAMYYCARHGRGYNG YEGAFDIWGQGTLVTVSS SEQ ID Lightchain variable DIQMTQSPSSLSASVGDRVTITCRASQGIGDSLAWY NO: 152 region ofAb89 QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPITFGQGTKVEIK SEQ ID Ab89 CDR-H1 NYWIG NO: 145 SEQID NO: 2 Ab89 CDR-H2 IIYPGDSDTRYSPSFQG SEQ ID NO: 3 Ab89 CDR-H3HGRGYNGYEGAFDI SEQ ID Ab89 CDR-L1 RASQGIGDSLA NO: 154 SEQ ID Ab89 CDR-L2DASNLET NO: 149 SEQ ID Ab89 CDR-L3 QQLNGYPIT NO: 155 SEQ ID NO: Heavychain QVQLVQSGAAVKKPGESLKISCKGSGYRFTSYWIG 161 variable regionWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTI amino acidSAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNG sequence of CK6 YEGAFDIWGQGTMVTVSSSEQ ID NO: Light chain variable AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ162 region amino acid QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTL sequence ofCK6 TISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID Ab77 CDR-H1 TYWIG NO: 163SEQ ID NO: 2 Ab77 CDR-H2 IIYPGDSDTRYSPSFQG SEQ ID NO: 3 Ab77 CDR-H3HGRGYNGYEGAFDI SEQ ID Ab77 CDR-L1 RASQGVISALA NO: 164 SEQ ID Ab77 CDR-L2DASILES NO: 165 SEQ ID Ab77 CDR-L3 QQFNSYPLT NO: 166 SEQ ID Ab79 CDR-H1TYWIG NO: 163 SEQ ID NO: 2 Ab79 CDR-H2 IIYPGDSDTRYSPSFQG SEQ ID NO: 3Ab79 CDR-H3 HGRGYNGYEGAFDI SEQ ID Ab79 CDR-L1 RASQGVGSALA NO: 167 SEQ IDAb79 CDR-L2 DASILES NO: 165 SEQ ID Ab79 CDR-L3 QQFNSYPLT NO: 166 SEQ IDAb81 CDR-H1 TYWIG NO: 163 SEQ ID NO: 2 Ab81 CDR-H2 IIYPGDSDTRYSPSFQG SEQID NO: 3 Ab81 CDR-H3 HGRGYNGYEGAFDI SEQ ID Ab81 CDR-L1 RASQGVISALA NO:164 SEQ ID Ab81 CDR-L2 DASTLES NO: 168 SEQ ID Ab81 CDR-L3 QQFNSYPLT NO:166 SEQ ID Heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 169constant region VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (Wild type (WT))SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID Heavy chainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 170 constant regionVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP with L234A, L235ASSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (LALA) mutationsCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC (mutations in bold)*VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID Heavy chainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 171 constant regionVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP with D265CSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT mutationCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV (mutation in bold)*VVCVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 172constant region VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP with H435ASSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT mutationCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV (mutation in bold)*VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID Heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 173constant region: VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP modified Fc regionSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT with L234A, L235A,CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC D265C mutationsVVVCVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY (mutations in bold)*NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID Heavy chainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP NO: 174 constant region:VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP modified Fc regionSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT with L234A, L235A,CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC D265C, H435AVVVCVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY mutationsNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI (mutations in bold)*EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQ KSLSLSPGK SEQ ID Ab85 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 175 heavy chainWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI sequence; constantSADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG region underlinedYEGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab85 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 176 heavy chainWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI sequence; constantSADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG region underlined;YEGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKST modified Fc regionSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF with L234A, L235APAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP mutationsSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF (mutations in bold)*LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab85 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 177 heavy chainWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI sequence: constantSADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG region underlined;YEGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKST modified Fc regionSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF with L234A, L235A,PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP D265C mutationsSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF (mutations in bold)*LFPPKPKDTLMISRTPEVTCVVVCVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab85 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIG NO: 178 heavy chainWVRQMPGKGLEWMAIINPRDSDTRYRPSFQGQVTI sequence (LALA-SADKSISTAYLQWSSLKASDTAMYYCARHGRGYEG D265C-H435AYEGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKST mutant); constantSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF region underlinedPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVCVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID Ab249 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 179 heavy chainVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA sequence; constantDKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE region underlinedGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab249 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 180 heavy chainVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA sequence; constantDKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE region underlinedGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG (LALA mutations)*GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab249 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 181 heavy chainVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA sequence; constantDKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE region underlinedGAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG (LALA-D265CGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV mutations)*LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVCVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Ab249 full lengthEVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGW NO: 182 heavy chainVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA sequence; constantDKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYE region underlined;GAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG (LALA-D265C-GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV H435A mutations)*LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVCVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID Light chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA NO: 183 constant regionKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID Ab85 full length DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWY NO: 184light chain; QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT constant regionLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIKRT underlinedVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID Ab249 light chain; DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWY NO: 185constant region QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT underlinedLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: Ab249 HC-CDR1 TSWIG 186 SEQ ID NO: Ab249 HC-CDR3HGLGYNGYEGAFDI 187 SEQ ID NO: Ab249 LC-CDR1 RASQGIGSALA 188 SEQ ID NO:Ab249 LC-CDR3 CQQLNGYPLT 189

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:
 1. An isolated anti-CD117 antibody, orantigen-binding fragment thereof, comprising (i) a heavy chain variableregion comprising a CDR1 domain comprising the amino acid sequence asset forth in SEQ ID NO: 145, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO:146, and a CDR3 domain comprising theamino acid sequence as set forth in SEQ ID NO: 147; and a light chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 148, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO:149, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 150; or(ii) a heavy chain variable region comprising the amino acid sequence asset forth in SEQ ID NO: 143, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO:
 144. 2.The anti-CD117 antibody, or antigen-binding fragment thereof, of claim1, wherein the antibody, or antigen-binding fragment thereof, bindsCD117 with a K_(D) of about 100 nM or less, about 90 nM or less, about80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM orless, about 40 nM or less, about 30 nM or less, about 20 nM or less,about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nMor less, about 2 nM or less, or about 1 nM or less as determined by aBio-Layer Interferometry (BLI) assay.
 3. The anti-CD117 antibody, orantigen-binding fragment thereof, of claim 1, wherein the antibody, orantigen-binding fragment thereof, is human.
 4. The anti-CD117 antibody,or antigen-binding fragment thereof, of claim 1, wherein the antibody isan intact antibody.
 5. The anti-CD117 antibody, or antigen-bindingfragment thereof, of claim 1, wherein the antibody is an IgG.
 6. Theanti-CD117 antibody, or antigen-binding fragment thereof, of claim 5,wherein the IgG is an IgG1 or an IgG4.
 7. The anti-CD117 antibody, orantigen-binding fragment thereof, of claim 1, wherein the antibody, orantigen-binding fragment thereof, is a monoclonal antibody.
 8. Theanti-CD117 antibody, or antigen-binding fragment thereof, of claim 1,comprising a heavy chain constant region having an amino acid sequenceas set forth in SEQ ID NO: 169 and/or a light chain constant regioncomprising an amino acid sequence as set forth in SEQ ID NO:
 183. 9. Theanti-CD117 antibody, or antigen-binding fragment thereof, of claim 1,comprising an Fc region comprising at least one amino acid substitutionselected from the group consisting of D265C, H435A, L234A, and L235A(numbering according to the EU index).
 10. The anti-CD117 antibody, orantigen-binding fragment thereof, of claim 1, comprising an Fc region,wherein the Fc region comprises amino acid substitutions D265C, L234A,and L235A (numbering according to the EU index).
 11. An intactanti-CD117 human antibody comprising a light chain comprising an aminoacid sequence as set forth in SEQ ID NO: 184, and a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO:
 178. 12.An intact anti-CD117 human antibody comprising a light chain comprisingan amino acid sequence as set forth in SEQ ID NO: 185, and a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO:
 182. 13.A pharmaceutical composition comprising the antibody, or antigen-bindingfragment thereof, of claim 1, and a pharmaceutically acceptable carrier.14. An antibody drug conjugate comprising an anti-CD117 antibodyconjugated to a cytotoxin via a linker, wherein the antibody comprisesthe antibody, or antigen-binding fragment thereof, of claim 1, whereinthe cytotoxin is selected from the group consisting of an amatoxin, apseudomonas exotoxin A, deBouganin, a diphtheria toxin, saporin, amaytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer.
 15. Theantibody drug conjugate of claim 14, wherein the amatoxin is selectedfrom the group consisting of α-amanitin, β-amanitin, γ-amanitin,ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, andproamanullin.
 16. An antibody drug conjugate (ADC) comprising anisolated anti-CD117 antibody, or antigen-binding fragment thereof,conjugated to a cytotoxin via a linker, wherein the antibody, orantigen-binding fragment therof, comprises (i) a heavy chain variableregion comprising a CDR1 domain comprising the amino acid sequence asset forth in SEQ ID NO: 145, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO:146, and a CDR3 domain comprising theamino acid sequence as set forth in SEQ ID NO: 147; and a light chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 148, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO:149, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 150; or(ii) a heavy chain variable region comprising the amino acid sequence asset forth in SEQ ID NO: 143, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO:
 144. 17.The ADC of claim 16, wherein the cytotoxin is selected from the groupconsisting of an amatoxin, an auristatin, a maytansine, a maytansinoid,a pyrrolobenzodiazepine, and a pyrrolobenzodiazepine dimer.
 18. Anantibody drug conjugate (ADC) represented by the formula Ab-Z-L-Am,wherein Ab is an antibody, or antigen-binding fragment thereof, thatbinds CD117, L is a linker, Z is a chemical moiety, and Am is anamatoxin, wherein the antibody, or antigen-binding fragment thereof,comprises (i) a heavy chain variable region comprising a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 145, aCDR2 domain comprising the amino acid sequence as set forth in SEQ IDNO:146, and a CDR3 domain comprising the amino acid sequence as setforth in SEQ ID NO: 147; and a light chain variable region comprising aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 148, a CDR2 domain comprising the amino acid sequence as set forthin SEQ ID NO:149, and a CDR3 domain comprising the amino acid sequenceas set forth in SEQ ID NO: 150; or (ii) a heavy chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 143, and alight chain variable region comprising the amino acid sequence as setforth in SEQ ID NO:
 144. 19. The ADC of claim 18, wherein the Am-L-Z isrepresented by formula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ areeach independently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH,NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), orOR_(D); X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof; and Z is a chemical moietyformed from a coupling reaction between a reactive substituent presenton L and a reactive substituent present within the antibody orantigen-binding fragment thereof, wherein Am comprises exactly one R_(C)substituent.
 20. The ADC of claim 18, wherein Am-L-Z is represented byformula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), when present, together with the oxygen atoms to whichthey are bound, combine to form an optionally substituted 5-memberedheterocycloalkyl group; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ areeach independently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH,NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), orOR_(D); X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, optionally substituted heteroarylene, a dipeptide,—(C═O)—, a peptide, or a combination thereof; and Z is a chemical moietyformed from a coupling reaction between a reactive substituent presenton L and a reactive substituent present within the antibody, orantigen-binding fragment thereof, wherein Am comprises exactly one R_(C)substituent.
 21. The ADC of claim 20, wherein L-Z is


22. The ADC of claim 18, wherein the antibody, or antigen-bindingfragment thereof, is conjugated to the amatoxin by way of a cysteineresidue in an Fc domain of the antibody, or antigen-binding fragmentthereof.
 23. The ADC of claim 22, wherein the cysteine residue isintroduced by way of a mutation in the Fc domain of the antibody, orantigen-binding fragment thereof.
 24. An antibody drug conjugate (ADC)represented by any one of the following:

wherein Ab is an anti-CD117 antibody or antigen-binding fragment thereofcomprising (i) a heavy chain variable region comprising a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 145, aCDR2 domain comprising the amino acid sequence as set forth in SEQ IDNO:146, and a CDR3 domain comprising the amino acid sequence as setforth in SEQ ID NO: 147; and comprising a light chain variable regioncomprising a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 148, a CDR2 domain comprising the amino acid sequence asset forth in SEQ ID NO:149, and a CDR3 domain comprising the amino acidsequence as set forth in SEQ ID NO: 150; or (ii) a heavy chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO:143, and a light chain variable region comprising the amino acidsequence as set forth in SEQ ID NO:
 144. 25. The ADC of claim 16,wherein the antibody, or antigen-binding fragment thereof, comprises aheavy chain constant region having an amino acid sequence as set forthin SEQ ID NO: 169 and/or a light chain constant region comprising anamino acid sequence as set forth in SEQ ID NO:
 183. 26. The ADC of claim16, wherein the antibody, or antigen-binding fragment thereof, comprisesan Fc region comprising at least one amino acid substitution selectedfrom the group consisting of D265C, H435A, L234A, and L235A (numberingaccording to the EU index).
 27. The ADC of claim 16, wherein theantibody, or antigen-binding fragment thereof, comprises an Fc region,wherein the Fc region comprises D265C, L234A, and L235A (numberingaccording to the EU index).
 28. The ADC of claim 16, wherein theantibody is an intact antibody and comprises a light chain comprising anamino acid sequence as set forth in SEQ ID NO: 184, and a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO:
 178. 29. Amethod of depleting a population of CD117+ cells in a human patient, themethod comprising administering to the patient an effective amount ofthe ADC of claim
 16. 30. A method of depleting a population of CD117+cells in a human patient in need of a hematopoietic stem celltransplant, the method comprising administering to the patient aneffective amount of the ADC of claim 16 prior to the patient receiving atransplant comprising hematopoietic stem cells.
 31. A method comprising:a. administering to a human patient the ADC of claim 16, in an amountsufficient to deplete a population of CD117+ cells in the patient; andb. subsequently administering to the patient a transplant comprisinghematopoietic stem cells.
 32. The method of claim 29, wherein thepatient is suffering from a disorder selected from the group consistingof a stem cell disorders hemoglobinopathy disorder, myelodysplasticdisorder, immunodeficiency disorder, a metabolic disorder, adenosinedeaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditarylymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storagediseases, thalassemia major, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, juvenile rheumatoid arthritis andcancer.
 33. A pharmaceutical composition comprising the ADC of claim 16,and a pharmaceutically acceptable carrier.
 34. An isolated anti-CD117antibody, or antigen-binding fragment thereof, comprising (i) a heavychain variable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 145, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 153, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 3; and alight chain variable region comprising a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 154, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 149, and aCDR3 domain comprising the amino acid sequence as set forth in SEQ IDNO: 155; (ii) a heavy chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 151, and a light chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO:152; (iii) a heavy chain variable region comprising a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 145, aCDR2 domain comprising the amino acid sequence as set forth in SEQ IDNO: 146, and a CDR3 domain comprising the amino acid sequence as setforth in SEQ ID NO: 147; and a light chain variable region comprising aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 157, a CDR2 domain comprising the amino acid sequence as set forthin SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence asset forth in SEQ ID NO: 155; (iv) a heavy chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 143, and alight chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 156; (v) a heavy chain variable region comprising aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 145, a CDR2 domain comprising the amino acid sequence as set forthin SEQ ID NO: 159, and a CDR3 domain comprising the amino acid sequenceas set forth in SEQ ID NO: 3; and a light chain variable regioncomprising a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 157, a CDR2 domain comprising the amino acid sequence asset forth in SEQ ID NO: 5, and a CDR3 domain comprising the amino acidsequence as set forth in SEQ ID NO: 155; (vi) a heavy chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO:158, and a light chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 156; (vii) a heavy chain variableregion comprising a CDR1 domain comprising the amino acid sequence asset forth in SEQ ID NO: 145, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 2, and a CDR3 domain comprising theamino acid sequence as set forth in SEQ ID NO: 3; and a light chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 154, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 149, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 155;(viii) a heavy chain variable region comprising the amino acid sequenceas set forth in SEQ ID NO: 160, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 152; (ix)a heavy chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 98, and a light chain variable region comprising theamino acid sequence as set forth in SEQ ID NO: 102; (x) a heavy chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 186, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 187; and alight chain variable region comprising a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 188, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 149, and aCDR3 domain comprising the amino acid sequence as set forth in SEQ IDNO: 189; (xi) a heavy chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 98, and a light chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO: 99;(xii) a heavy chain variable region comprising the amino acid sequenceas set forth in SEQ ID NO: 7, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 99; (xiii)a heavy chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 7, and a light chain variable region comprising theamino acid sequence as set forth in SEQ ID NO: 100; or (xiv) a heavychain variable region comprising the amino acid sequence as set forth inSEQ ID NO: 98, and a light chain variable region comprising the aminoacid sequence as set forth in SEQ ID NO:
 101. 35. An antibody drugconjugate (ADC) comprising an isolated anti-CD117 antibody conjugated toa cytotoxin via a linker, wherein the antibody comprises (i) a heavychain variable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 145, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 153, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 3; and alight chain variable region comprising a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 154, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 149, and aCDR3 domain comprising the amino acid sequence as set forth in SEQ IDNO: 155; (ii) a heavy chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 151, and a light chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO:152; (iii) a heavy chain variable region comprising a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 145, aCDR2 domain comprising the amino acid sequence as set forth in SEQ IDNO: 146, and a CDR3 domain comprising the amino acid sequence as setforth in SEQ ID NO: 147; and a light chain variable region comprising aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 157, a CDR2 domain comprising the amino acid sequence as set forthin SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence asset forth in SEQ ID NO: 155; (iv) a heavy chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 143, and alight chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 156; (v) a heavy chain variable region comprising aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 145, a CDR2 domain comprising the amino acid sequence as set forthin SEQ ID NO: 159, and a CDR3 domain comprising the amino acid sequenceas set forth in SEQ ID NO: 3; and a light chain variable regioncomprising a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 157, a CDR2 domain comprising the amino acid sequence asset forth in SEQ ID NO: 5, and a CDR3 domain comprising the amino acidsequence as set forth in SEQ ID NO: 155; (vi) a heavy chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO:158, and a light chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 156; (vii) a heavy chain variableregion comprising a CDR1 domain comprising the amino acid sequence asset forth in SEQ ID NO: 145, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 2, and a CDR3 domain comprising theamino acid sequence as set forth in SEQ ID NO: 3; and a light chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 154, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 149, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 155;(viii) a heavy chain variable region comprising the amino acid sequenceas set forth in SEQ ID NO: 160, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 152; (ix)a heavy chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 98, and a light chain variable region comprising theamino acid sequence as set forth in SEQ ID NO: 102; (x) a heavy chainvariable region comprising a CDR1 domain comprising the amino acidsequence as set forth in SEQ ID NO: 186, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 187; and alight chain variable region comprising a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 188, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 149, and aCDR3 domain comprising the amino acid sequence as set forth in SEQ IDNO: 189; (xi) a heavy chain variable region comprising the amino acidsequence as set forth in SEQ ID NO: 98, and a light chain variableregion comprising the amino acid sequence as set forth in SEQ ID NO: 99;(xii) a heavy chain variable region comprising the amino acid sequenceas set forth in SEQ ID NO: 7, and a light chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO: 99; (xiii)a heavy chain variable region comprising the amino acid sequence as setforth in SEQ ID NO: 7, and a light chain variable region comprising theamino acid sequence as set forth in SEQ ID NO: 100; or (xiv) a heavychain variable region comprising the amino acid sequence as set forth inSEQ ID NO: 98, and a light chain variable region comprising the aminoacid sequence as set forth in SEQ ID NO:
 101. 36. The antibody drugconjugate of claim 35, wherein the cytotoxin is selected from the groupconsisting of an amatoxin, a pseudomonas exotoxin A, deBouganin, adiphtheria toxin, saporin, a maytansine, a maytansinoid, an auristatin,an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer.
 37. Apharmaceutical composition comprising the intact anti-CD117 antibody ofclaim 11 or 12, and a pharmaceutically acceptable carrier.
 38. Apharmaceutical composition comprising the ADC of claim 18, 24, or 35,and a pharmaceutically acceptable carrier.